CN102985552B - For detecting the method and composition of genetic material - Google Patents
For detecting the method and composition of genetic material Download PDFInfo
- Publication number
- CN102985552B CN102985552B CN201080062146.9A CN201080062146A CN102985552B CN 102985552 B CN102985552 B CN 102985552B CN 201080062146 A CN201080062146 A CN 201080062146A CN 102985552 B CN102985552 B CN 102985552B
- Authority
- CN
- China
- Prior art keywords
- probe
- droplet
- target polynucleotide
- linking
- target
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
- C12Q1/6855—Ligating adaptors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides the composition for detecting target polynucleotide copy number difference and method.In some cases, when initial sample is maternal tissue (such as, blood, blood plasma), method and composition provided herein can be used for diagnosing fetal genetic abnormality.Though the technology of described method and materials application allows detection polynucleotide copies number small but the difference of statistically significant.
Description
the cross reference of related application
The application requires the U.S. Provisional Application numbers 61/264,591 of acquisition application on November 25th, 2009 according to United States Code the 35th section the 119th (e) article, the U.S. Provisional Application numbers 61/309,837 of application on March 2nd, 2010, the U.S. Provisional Application numbers 61/309,845 of application on March 2nd, 2010, the U.S. Provisional Application numbers 61/317,635 of application on March 25th, 2010, the U.S. Provisional Application numbers 61/317,639 of application on March 25th, 2010, the U.S. Provisional Application numbers 61/317,684 of application on March 25th, 2010, the U.S. Provisional Application number 61/341,065 of application on March 25th, 2010, the U.S. Provisional Application numbers 61/341,218 of application on March 25th, 2010, the U.S. Provisional Application numbers 61/380,981 of application on September 8th, 2010, the U.S. Provisional Application numbers 61/409,106 of application on November 1st, 2010, the U.S. Provisional Application numbers 61/409,473 of application on November 2nd, 2010, the interests of the U.S. Provisional Application numbers 61/410,769 of application on November 5th, 2010, wherein each application entirety is quoted and is incorporated to herein.
Background technology
Fetus dysploidy is the distortion of chromosome number, normally causes because the reduction division during ovum or spermatogenesis is not separated; But some dysploidy is if trisomy 8 is more generally because mitotic division after zygote is separated (Nicolaidis and Petersen (1998) HumanReproduction13:313-319) caused.Such distortion comprises minimizing and the increase of normal chromosomal number, and can relate to euchromosome and sex chromosome.An example of minimizing property dysploidy is Turner syndrome, and its characteristic feature there is single X sex chromosome.The example that chromosome number increases comprises mongolism (karyomit(e) trisomy 21), Patau's syndrome (karyomit(e) 13 3 body), Edward's syndrome (karyomit(e) 18 3 body) and klinefelter syndrome (Kleinfelter ' ssyndrome) (heterosomal XXY tri-body).Dysploidy causes significant health and nerve injury usually, and this causes the affected individuals of very high per-cent can not survive to growing up.In fact, except 13,18 or 21, other chromosomal euchromosome dysploidy fetus is usually dead in uterus.But, some dysploidy, as klinefelter syndrome, the phenotype shown is very not obvious, and those grownups usually reached maturity as having Fertility as XXY and XXX influencer by other trisomy.In some cases, the part dysploidy of the copy number exception of a karyomit(e) part is caused may to cause due to unbalanced separation.
Invasive detection (such as by amniocentesis or chorionic villus sampling (CVS)) is adopted fetus to be carried out to the antenatal diagnosis of dysploidy, this operates the relevant (D ' Alton of relevant risk with the miscarriage of 0.5%-2%, M.E., (1994) SeminPerinatol18:140-62; CaugheyAB (2006) ObstetGynecol108:612-6).
Another obstacle of accurate examination fetus dysploidy is that in mother's blood plasma, foetal DNA concentration is lower, especially pregnant age comparatively early time.Single channel or rudimentary multi-way detecting method can not provide enough target number to distinguish orthoploidy fetus and dysploidy fetus (such as karyomit(e) trisomy 21).Usually, this area also needs the method and composition for detecting copy number variation in biological sample (not necessarily coming from mother's blood).
Summary of the invention
The disclosure provides the method and composition of the copy number for detecting target polynucleotide in genetic material colony.Can utilize to distribute and target polynucleotide is sub-divided into multiple reaction volume.In some cases, the probe for target polynucleotide subdivided into multiple reaction volume.
In some cases, described method comprises the steps: that the first linking probe is combined with the first target polynucleotide by a.; B. the second linking probe is combined with the second target polynucleotide; C. make described first and second linking probes carry out ligation and connect product to obtain one or more; D. described one or more being connected product is assigned in two or more subregions; E. the sequence in amplification one or more connection products described is to obtain amplified production; F. the number of the described subregion comprising described amplified production is determined; And g. calculates the copy number of described first target polynucleotide.In some cases, target polynucleotide is not assigned in two or more subregions described.
Subregion can comprise polytype subregion, comprises solid subregion (i.e. hole, pipe etc.) and fluid subregion (such as oil phase is as the water-based droplet in the mixture of the water-based droplet in oil-continuous phase or at least two kinds of Immiscible fluids).Subregion also can be stable or instability.Such as, in some cases, during amplification procedure, two or more subregions described keep complete substantially.In some cases, subregion is the water-based droplet in oil phase, and described water-based is little drop in the amplified reaction of present method during basic keep complete.When evaluating subregion and whether there is one or more target polynucleotide the probe of described polynucleotide (or for), during determining step, described subregion (such as described water-based droplet) also can keep complete substantially.Described subregion may comprise from the initial amplified reaction of described connection product.
First and second linking probes can in conjunction with (or being designed to combine) multiple target polynucleotide; Usually, the first linking probe is in conjunction with the first target polynucleotide, and the second linking probe is in conjunction with the second polynucleotide.In some cases, the first and second linking probes are designed to separately in conjunction with described first target polynucleotide.In other cases, described first linking probe is in conjunction with the first target polynucleotide, and the sequence of this first target polynucleotide is different from the sequence of described second target polynucleotide.In some cases, the first linking probe is designed to be combined in polynucleotide sequence conservative between kind of interior individuality.In some cases, the first linking probe is designed to be combined in two or more not of the same race conservative polynucleotide sequences.In some cases, linking probe is in conjunction with chromosomal non-polymorphic regions.
In some embodiments, described method comprises many linking probes is connected to described first target polynucleotide.Such as, described method can comprise at least 4 linking probes are incorporated into described first target polynucleotide.In other cases, in method provided herein, use at least 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,100,200,500,1000,5000,10,000,20,000,30,000,40,000,50,000,60,000,70,000,100,000,2,000,000,3,000,000,4,000,000,5,000,000,6,000,000,7,000,000,8,000,000,9,000, article 000 or 10,000,000, linking probe.Usually, one or more described linking probes is incorporated into different polynucleotide (such as, coloured differently body, identical intrachromosomal different zones).In some cases, in present method and composition, use multiple first linking probe (such as target linking probe), and use multiple second linking probe (such as with reference to linking probe).The method can comprise further at least 4 linking probes are incorporated into described first target polynucleotide, and at least 4 linking probes are incorporated into described second target polynucleotide.In some cases, at least 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,100,200,500,1000,5000,10,000,20,000,30,000,40,000,50,000,60,000,70,000,100,000,2,000,000,3,000,000,4,000,000,5,000,000,6,000,000,7,000,000,8,000,000,9,000, article 000 or 10,000,000, linking probe is incorporated into described first or described second target polynucleotide.
First linking probe can in conjunction with the first area in (or being designed to combine) described first target polynucleotide, and described second linking probe can in conjunction with the second area in (or being designed to combine) described first target polynucleotide, and wherein said first and second regions do not have identical sequence.
First target polynucleotide is usually not identical with described second target polynucleotide.In some cases, the first target polynucleotide is identical with described second target polynucleotide.In some instances, described first target polynucleotide is test chromosome, and described second target polynucleotide is with reference to karyomit(e).The example of test chromosome includes but not limited to: karyomit(e) 21, karyomit(e) 13, karyomit(e) 18 and X chromosome.Described test chromosome also can be selected from karyomit(e) 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, X and Y.First target polynucleotide can be chromosomal section, as the chromosomal section (this karyomit(e) or this section can with fetus dysploidy be correlated with) relevant to fetus dysploidy.
Method and composition provided herein is usually directed to be connected with himself by probe, be linked together by two probes, and/or the connection product of described ligation.Described ligation can cause the connection in 3 ' region of 5 ' region of described first linking probe and described first linking probe, to obtain the connection product of ring-type.In some cases, ligation causes the connection in 3 ' region of 5 ' region of described first linking probe and described second linking probe, to obtain the linearly connected product at least partially comprising described first and second linking probes.Described 5 ' region and the described 3 ' region of described first linking probe can combine the flanking sequence in (or being designed to combine) described first target polynucleotide separately.Described flanking sequence is separated by 0 Nucleotide.Described 5 ' the region of described first linking probe and described 3 ' region can in conjunction with or be designed in conjunction with the contiguous sequence in described first target polynucleotide.Described contiguous sequence can be separated by least 1 Nucleotide.In some cases, described contiguous sequence is separated by the breach of at least 5,10,20,30,40,50,100,200,300,400 or 500 Nucleotide.
Described ligation can comprise the gap fill reaction of template-driven further, to introduce Nucleotide in (or the described second linking probe) described 5 ' region of described first linking probe and described 3 ' interregional breach.
Linking probe can comprise the site that can digestedly cut.Such as, the site can digestedly cut can comprise one or more uridylics.In some cases, described uridylic can be separated by other Nucleotide.The site that can digestedly cut can comprise restriction site.First linking probe can be particular type, as molecular inversion probes, snap close probe (padlockprobe), linearly connected probe etc.
Method provided herein can comprise further carries out enzyme reaction to remove linear polynucleotides or single stranded polynucleotide or double-stranded polynucleotide.Such as, exonuclease (such as, ExoI, II and/or III) can be used in method described herein.Exonucleolytic ferment treatment often removes all or unconjugated linking probe of significant quantity from sample volume.
Probe provided herein can with signal reagent coupling.Described first linking probe can be coupled to the first signal reagent, and the second linking probe is coupled to second signal reagent.Usually, many the first and second linking probes are like this used in method and composition herein, wherein said probe conjugate is in identical signal reagent (such as, identical fluorophore) or different signal reagent (such as, the fluorophore of different colours).Described first signal reagent can be the fluorescent marker of the first color, and described second signal reagent can be the fluorescent marker of the second color.
Detection linking probe is also usually the step in method provided herein.Described method can comprise detecting and has described second linking probe that described first linking probe of the first signal reagent and detection have second signal reagent.Described first linking probe can comprise first group of linking probe, every bar probe in wherein said group is for the different zones of the first chromosome, wherein said second linking probe comprises second group of linking probe, and the every bar probe in wherein said group is for the second chromosomal different zones.In some cases, described first target polynucleotide is test chromosome, and described second target polynucleotide is with reference to karyomit(e).In some cases, described first and second linking probes are coupled to identical color.
Method and composition provided herein also can relate to the method for the copy number detecting target polynucleotide in genetic material colony, and the method comprises: the first linking probe is combined with the first target polynucleotide by a.; B. the second linking probe is combined with the second target polynucleotide; C. make described first and second linking probes carry out ligation and connect product to obtain one or more; D. described one or more being connected product is assigned in two or more water-based droplets in oil-continuous phase; E. the sequence in amplification one or more connection products described is to obtain amplified production; F. the number of two or more water-based droplets described comprising described amplified production is determined; And g. calculates the copy number of described target polynucleotide based on described number.In some cases, described target polynucleotide is not assigned in two or more water-based droplets described.In some cases, described target polynucleotide is not amplified.In some cases, or during described amplification or determining step, two or more water-based droplets described keep complete substantially.
In some cases, two or more water-based droplet average packets described contain more than a linking probe, and described method comprises the mean number calculating target linking probe in each water-based droplet with algorithm further.Two or more water-based droplets described may more than 4,000 droplet.In some cases, two or more water-based droplets described may more than 1,000,10,000,20,000,50,000,100,000,200,000,500,000,1,000,000 or 5,000,000 droplet.
In some cases, described droplet is present in single chamber with the density of high droplet number/ml.Described density can higher than 100,000 water-based droplet/ml.In single chamber, the example of droplet density comprises: 10,000 droplet/ml, 100,000 droplet/ml, 200,000 droplet/ml, 300,000 droplet/ml, 400,000 droplet/ml, 500,000 droplet/ml, 600,000 droplet/ml, 700,000 droplet/ml, 800,000 droplet/ml, 900,000 droplet/ml or 1,000,000 droplet/ml.Droplet for any method provided herein or composition can be single dispersing droplet.Droplet on average can have the diameter of 50nm-300 μm.In some embodiments, droplet diameter can be on average about 0.001,0.01,0.05,0.1,1,5,10,20,30,40,50,60,70,80,100,120,130,140,150,160,180,200,300,400 or 500 micron.In some cases, droplet does not comprise a large amount of microballons being coupled to oligonucleotide.
Water-based droplet can be present in flow of oil or oil phase.Oil phase can comprise anionic fluorosurfactants, and the ammonium salt of anionic fluorosurfactants, as Krytox
tM.Krytox can be selected from the morpholino derivative of KrytoxAS, KrytoxFSH and KrytoxFSH.Oil phase can comprise fluorinated oil.
Method provided herein (such as, using droplet to detect copy number) can be used for detection and comprises and be less than 1, intragroup described first target polynucleotide of genetic material of described first target polynucleotide of 000 copy.In some cases, two or more water-based droplet average packets described contain more than a linking probe, and described method comprises the mean number calculating target linking probe in each water-based droplet with algorithm further.
Droplet can comprise the first target polynucleotide of the chromosome segment relevant to inherited disease.Droplet can comprise the linking probe (such as snap close probe, molecular inversion probes, joint detection reaction (LDR) probe etc.) of particular type.Linking probe can carry out ligation, is connected in 5 ' of linking probe region with 3 ' region of linking probe.
Method and composition provided herein also can relate to the method detecting fetus genetic situation, comprising: a. obtains and comprises mother of target polynucleotide and the mixture of fetal genetic material; B. described mixture and the target oligonucleotide in conjunction with described target polynucleotide are combined; C. be sub-divided in reaction volume by described target oligonucleotide, at least one in wherein said reaction volume does not comprise target polynucleotide and target oligonucleotide; D. in described reaction volume, amplified reaction is carried out; E. the existence of described target polynucleotide or described target oligonucleotide in described reaction volume is detected; And f. determines that the relative level of target polynucleotide described in described mixture is to detect the hereditary conditions of fetus.
Reaction volume can be the water-based droplet in oil-continuous phase.Target oligonucleotide can comprise one or more primer pair, linking probe, molecular inversion probes, joint detection reaction (LDR) probe, snap close probe and arbitrary combination thereof.Reaction volume can comprise the target oligonucleotide of average more than one copy and/or the target polynucleotide of average more than one copy.Described reaction volume can comprise further for the primer with reference to polynucleotide.In some cases, described reaction volume comprises further for the linking probe with reference to polynucleotide.
In some embodiments, linking probe increases in described reaction volume.
The fetal genetic material used in the method for detection fetus genetic state can derive from the cell sample for the preconcentration of fetal genetic material selectivity.But in some embodiments, the fetal genetic material used in the method for detection fetus genetic state does not derive from the cell sample for the preconcentration of fetal genetic material selectivity.
Target polynucleotide may be selected from karyomit(e) 18,13,21 and X or be selected from karyomit(e) 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, in the karyomit(e) of X or Y.
In some cases, described reaction volume is the water-based droplet in oil phase, described target oligonucleotide is linking probe, and the droplet number that described determining step comprises the amplified production by comprising described linking probe compares with the droplet number comprised for the amplified production of the linking probe with reference to polynucleotide.In some cases, described is abnormal irrelevant chromosomal regions with fetus genetic with reference to polynucleotide.
As used in the method and composition that provides herein, described target oligonucleotide can be the linking probe becoming ring-type after hybridizing with target polynucleotide through being connected.
The disclosure also provides the method for composition microcapsule compositions as described in microcapsule compositions and use.In some cases, composition is the microcapsule comprising linking probe, and wherein said microcapsule obtain as follows: the target polynucleotide of many linking probes in genetic material is optionally combined by a.; B. 5 ' end of the linking probe combined described at least one is connected with same or other the 3 ' end of linking probe of combination, thus acquisition at least one connection product; C. the aqueous solution introducing of product is connected for generation of in the device of droplet by comprising described at least one; D. produce the water-based droplet comprising described at least one and connect product with described device, wherein said water-based is little to be dropped in immiscible fluid; And described droplet is converted into the microcapsule comprising solid phase outside by e..In some cases, described conversion is included in more than 50 DEG C heating or heats more than 70 DEG C.Immiscible liquid (such as oil) can comprise fluorinated surfactant.In some cases, aqueous phase comprises fluorinated surfactant.Oil can be fluorocarbon oil.Oil phase can comprise anion surfactant.Oil phase can comprise Krytox ammonium.In some cases, described microcapsule do not comprise the microballon being incorporated into oligonucleotide.Described microcapsule can substantially keep complete at higher than the temperature of 70 DEG C.Microcapsule can comprise the linking probe that optionally can combine the target polynucleotide relevant to inherited disease.In some cases, described linking probe can optionally combine the target polynucleotide relevant to fetus dysploidy.In some cases, described genetic targets mark is in the karyomit(e) being selected from karyomit(e) 21, karyomit(e) 13, karyomit(e) 18 and X chromosome.In some cases, described genetic targets mark in karyomit(e) 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, in X or Y.
Microcapsule can comprise one or more of described linking probe (such as snap close probe, molecular inversion probes, joint detection reaction (LDR) probe, cycling probe etc.).Microcapsule can comprise the linear probe by the probe linearizing of previous cyclisation being obtained after ligation.In some cases, microcapsule comprise the linking probe of cyclisation.In some cases, microcapsule comprise the linear product of joint detection reaction (LDR).
Composition provided herein and method also can relate to the water-in-oil-type mixture comprising two or more water-based droplets, at least one in two or more water-based droplets wherein said comprises the first linking probe for the first target polynucleotide, and at least one in two or more water-based droplets described comprises the second linking probe for the second target polynucleotide.Described first target polynucleotide and described second target polynucleotide can be identical molecules, or have same sequence or the differing molecular of structure, or have the differing molecular of different sequence or structure.In some cases, described first target polynucleotide has the sequence different from the sequence of described second target polynucleotide.In some cases, described first target polynucleotide has the sequence identical with described second target polynucleotide.Described first target polynucleotide can comprise the first area in genomic segment, and described second target polynucleotide can comprise the second area in described genomic segment, and wherein said first area does not have the sequence identical with described second area.
In some cases, described water-in-oil-type mixture comprises Krytox ammonium surfactant further.Described Krytox tensio-active agent can be present in the concentration of at least 0.01% in the oil phase of described mixture.
In some cases, the linking probe that described mixture comprises is the linear product that cycling probe is cut through enzyme.In some cases, described mixture comprises the probe of cyclisation itself.In some cases, linking probe can comprise restriction enzyme site, can carry out as uracil-N-glycosylase or restricted enzymatic enzyme are cut at this place.
The present invention includes the method for the target sequence in mother and fetal genetic material mixture being carried out to Difference test, comprise the steps: a) to obtain the maternal tissue comprising mother and fetal genetic material; B) be assigned in discrete samples by genetic material, every increment product average packet is containing being no more than about target sequence/sample, and wherein discrete samples comprises one group of primer for known target sequence and/or one group of reference primer for known canonical sequence; C) amplified reaction is carried out; D) existence of target or canonical sequence in discrete samples is detected; And the ratio of e) relatively detected target sequence and detected canonical sequence, to determine the measures of dispersion of target sequence.Described method can comprise the ratio of relatively more detected target sequence and detected canonical sequence further to determine the step of the measures of dispersion of target sequence, and wherein detected target sequence and the difference of detected canonical sequence show that fetus genetic extremely.In some embodiments, described method detects the method for fetus dysploidy.In some cases, described target sequence is the marker of dysploidy, and described canonical sequence is diploid in mother and fetal genetic material.Maternal tissue can be the peripheral blood of mother, blood plasma or serum, or other tissue described herein.In some embodiments, response sample is the aqueous phase in emulsion.In some cases, the existence detecting target or canonical sequence comprises it further and has fluorescently-labeled nucleic acid hybridization in situ.In some cases, the number of response sample is at least about 10,000.In some cases, with the primer sets repeating step b for different target sequence)-e).In some cases, reaction volume comprises more than one primer sets, and wherein each primer sets is for particular target sequence.In some cases, reaction volume comprises more than one with reference to primer sets, and wherein each primer sets is for specific canonical sequence.The example of operable primer sets comprises the primer sets of specificity for human chromosome 21, human chromosome 18, human chromosome 13 or human chromosome X.In some cases, when the ratio of the target sequence detected and canonical sequence is greater than 1, dysploidy is detected.In some cases, when the ratio of the target sequence detected and canonical sequence is less than 1, dysploidy is detected.In some cases, target sequence be CFTR, Factor IX (F8 gene), beta globin, hemochromatosis, G6PD, multiple neurofibromatosis, GAPDH, amyloid beta or gene encoding for pyruvate kinase at least partially.
quote and be incorporated to
The all publications mentioned in this specification sheets and patent application are intactly quoted all herein and are incorporated to, and its degree is equal to each independent publication or patent application by specific and quote individually and be incorporated to.
Accompanying drawing explanation
Illustrate new feature of the present invention in the appended claims.By reference to the as detailed below and the accompanying drawing feature and advantage that the present invention may be better understood that describe illustrative embodiment (wherein have employed principle of the present invention), its accompanying drawing is as follows:
Fig. 1 general schematic view is that the step can taked by using droplet digital pcr and linking probe to detect the copy number variation in Patient Sample A is described.
Fig. 2 schematic diagram is an example of the step that can be used for detecting karyomit(e) (or its part) number change in sample.
Fig. 3 schema shows the illustrative methods for diagnosing fetal dysploidy.
Fig. 4 schematic diagram uses molecular inversion probes (MIP) to detect two kinds of hereditary targets.
Fig. 5 shows MIP method multiplex to strengthen the sensitivity of hereditary target detection.
Fig. 6 shows without the need to cutting, uses universal primer and general probe to detect the two color system of the nucleic acid in droplet.
Fig. 7 shows the PCR using joint detection reaction (LDR) and carry out in droplet subsequently, by two kinds of color detection, two kinds of genetic targets target schemes.
The LDR-PCR that the oligonucleotide that Fig. 8 shows use multiplex carries out in droplet is to strengthen the sensitivity detected.
Fig. 9 shows the computer for showing, storing, retrieve or calculate data or the result obtained by method and composition described herein.
Figure 10 shows the dependency between the input copy number of template DNA and positive droplet number (or counting), and for test sample, compares the sensitivity that the heavy MIP (upper figure) of 3-strengthens when using 12-heavy MIP (figure below).
Figure 11 shows for reference sample, and positive droplet number (or counting) is relative to template copy numbers.
Figure 12 shows the hybridization efficiency of the MIP multiplex for different templates copy number and different levels.
Figure 13 shows 24 MIP (SEQIDNO:1-24) for different zones in karyomit(e) 1.
Figure 14 shows 24 MIP (SEQIDNO:25-48) for different zones in karyomit(e) 21.
Figure 15 shows heavy MIP group (SEQIDNO:49-51) of 3-for different zones in karyomit(e) 1 and heavy MIP group (SEQIDNO:52-63) of 12-for different zones in karyomit(e) 1.
Figure 16 shows heavy MIP group (SEQIDNO:64-66) of 3-for different zones in karyomit(e) 21 and heavy MIP group (SEQIDNO:67-78) of 12-for different zones in karyomit(e) 21.
Figure 17 shows the exemplary universal primer and probe (SEQIDNO:79-82) that detect for MIP.
Detailed Description Of The Invention
Overview
The disclosure provides the method and composition for detecting heritable variation in biological sample.In some cases, the disclosure provides the method and composition of the copy number for detecting target polynucleotide in biological sample (such as, karyomit(e), chromosome segment, gene etc.).In some cases, the method and composition for detecting genetic mutation and/or single nucleotide polymorphism (SNP) in biological sample is also provided.
The disclosure additionally provides composition for detection resources fetus dysploidy or other genetic abnormality in the biological sample of maternal tissue and method.Such biological sample generally includes the mixture of mother and fetal nucleic acid (such as DNA, RNA).Dysploidy is chromosome abnormalty, refers to that the copy number of karyomit(e) or its fragment or its part is abnormal.Method described herein and some technology of materials application, these technology are used for analysis bank tissue samples as a large amount of nucleic acid comprised in blood (whole blood or peripheral blood), serum or blood plasma, these tissue samples comprise mother and foetal DNA (and/or DNA fragmentation) mixture, and permission detects the minute differences between target and reference DNA level that can indicate fetus dysploidy.
Copy number variation (CNV) used herein refers to acquisition or the loss of the section of genetic material.In the mankind, there is a large amount of CNV regions, between general groups, have genetic diversity widely.CNV also works in many human genetic diseases.The method especially can be used for the sex chromosomal abnormality detecting transposition, increase, amplification, transversion, reversion, dysploidy, polyploidy, monosomy, trisomy, trisomy 21,13 3 bodies, 14 3 bodies, 15 3 bodies, trisomy 16,18 3 bodies, 22 3 bodies, triploidy, tetraploidy and include but not limited to XO, XXY, XYY and XXX.Present method also provides the sequence for determining foetal DNA and identifies the noninvasive technology of the sudden change in foetal DNA.
The disclosure provides the means for detecting CNV, heritable variation and/or fetus dysploidy, such as by using digital pcr (such as, droplet digital pcr) and the probe of specialization, be commonly referred to linking probe herein (such as, molecular inversion probes and other probe), can link together directly or indirectly when hybridizing with target polynucleotide.In the method provided herein, comprise the sample of Target nucleotides or be assigned in multiple subregion (such as, droplet) for the probe of described Target nucleotides.Then at subregion (such as, droplet) carry out thermal cycle reaction to promote to comprise Target nucleotides or react for the PCR in the subregion of the probe of described Target nucleotides, thus produce the product (such as, DNA, RNA of amplification or other nucleic acid) of amplification.
In some embodiments, single probe is connected (such as, molecular inversion probes) at its end after hybridizing with target polynucleotide.In other embodiments, linking probe two molecules separated that can link together after being included in and hybridizing with target polynucleotide.
Composition described herein comprises and comprises the composition of the nucleic acid probe (such as, molecular inversion probes, linking probe etc.) of a type containing two or more Immiscible fluids as the mixture of oil and water.In other cases, composition described herein comprises the microcapsule of the nucleic acid probe (such as, molecular inversion probes, linking probe etc.) comprising a type.Such microcapsule may resist merging, especially when high temperature, therefore enable amplified reaction carry out with unusual high-density (such as, the stoichiometric number of each unit volume).
Fig. 1 provides a general schematic view, shows the step for taking by using droplet digital pcr (ddPCR) and linking probe to detect the copy number variation in Patient Sample A.The sample (101) of genomic nucleic acids (such as, genomic dna or RNA) is extracted from the sample obtained by patient (101).Probe (linking probe as described here) is hybridized (103) with the target nucleotide sequence in Patient Sample A; After hybridization, probe links together (104), and then sample optionally carries out ferment treatment (such as, exonuclease) to decompose genomic nucleic acids and the remaining probe (105) be not connected.Then PCR reactive component (such as, primer, fluorescent detection probe, polysaccharase, dNTP etc.) is added sample (106), then it is assigned to (107) in multiple droplet.After forming droplet, droplet carries out thermal cycling with the probe (108) increased in sample.Then determine number (109) that is positive and negative droplet, it is used to the Relative copy number determining target polynucleotide.Although Fig. 1 depicts droplet as distribution means, also can use other distribution means known in the art, such as, distribute between the hole in nanometer or microfluidic device.Further, although Fig. 1 depicts detect copy number variation, also other hereditary conditions can be detected.
In sample, the detection of copy number may relate to detection chromosome abnormalty, comprises dysploidy.The general schematic view of Fig. 2 is to identify fetus dysploidy in maternal sample and the step that can take.Primary tissue samples (201) comprises the mixture of mother and foetal DNA.Extract DNA, mix with for karyomit(e) 1 (202) (with reference to karyomit(e)) and the probe of karyomit(e) 21 (test chromosome) (203).Probe is combined with hereditary target, is then assigned to multiple subregion (204).Probe in sense partitions, test chromosome will be comprised (such as, karyomit(e) 21) the number of subregion compare (205) with the number of subregion comprised with reference to karyomit(e) (such as, karyomit(e) 1), calculate the Relative copy number of karyomit(e) 21 subsequently.
The disclosure provides for hereditary conditions to maternal tissue (such as, blood, serum or blood plasma) analyze, wherein analyze in maternal tissue the fetus that mixes with mother body D NA fetus sudden change or genetic abnormality to be distinguished mutually with the background of mother body D NA.Utilize the combination of step, the DNA sample containing mother and foetal DNA (or RNA) can be analyzed, to measure the relative concentration of the acellular DNA sequence dna in peripheral circulation.This concentration difference can be used distinguish and represent the hereditary conditions existed in the smaller portions DNA of foetal DNA.
The method can adopt numerical analysis, and the DNA wherein in sample is translated into the probe of multiple connection, and these probes are allocated to nominal single linking probe molecule in reaction volume, to generate sample mixture.Such as, reaction volume can be droplet, as the aqueous phase droplet disperseed in immiscible liquid, as U.S. Patent number 7, described in 041,481 (they are incorporated to by intactly quoting herein).Each reaction volume may within it only be distributed with less than a target (such as, target polynucleotide, targeted probes or other target molecules) or one or more target (such as, target polynucleotide, targeted probes or other targeted molecular).Can detect the target molecules in each reaction volume, detect preferably as the target sequence be amplified, described detection can comprise and quantizes (or quantitatively) to the starting copy number of target sequence, i.e. 0,1,2,3 copy etc.The exception that canonical sequence can be used to distinguish in target sequence increases, such as trisomy.Therefore, can carry out Difference test relative to canonical sequence to target sequence, there is fetus dysploidy in the instruction of this Difference test result.Canonical sequence is parental sequences not necessarily.
In addition, the method also can adopt far-ranging method to catch directly or indirectly and to detect fetal genetic material.A kind of method described herein comprises following combination: use molecular inversion probes (MIP) (or other oligonucleotide probe) instead of pair of primers in conjunction with genomic dna, step subsequently comprises the hybridization step be combined by the complementary sequence of MIP probe in target polynucleotide, make the ligation step of the circularizing probes of combination, digest the exonuclease treatment step of remaining non-cyclizing MIP probe, optional treatment step, wherein uses enzyme if uracil-N-glycosylase is by the probe linearizing of cyclisation; Allocation step, its cyclized by treatment probe or linearizing probe (it had been previously ring-type) are assigned with or are sub-divided in two or more subregion (such as, droplet); Amplification step subsequently relates to the sequence by droplet digital pcr amplification oligonucleotide probe uniqueness.
In some cases, use multiplex MIP (or other oligonucleotide) to improve the sensitivity of detection herein.Such as, can use one group of two or more MIP, each wherein in these MIP combines from the different sequences on phase homologous chromosomes (such as karyomit(e) 21).In some cases, identify that multiple MIP of such as target and canonical sequence can carry out Difference test with the fluorophore of different colours during increasing.In some cases, single linear probe is combined with genomic dna, and ligation subsequently produces the molecule of ring-type.In other cases, two linear probes are in conjunction with the adjacent area of genomic dna, and ligation subsequently produces can react the join dependency molecule be detected in (LDR) in joint detection.
Term used herein connects and refers to covalent linkage between two or more nucleic acid such as oligonucleotide and/or polynucleotide or connection.Usually, connection can comprise polynucleotide (such as linking probe) 5 ' end with the 3 ' end of another polynucleotide (such as linking probe) or be connected with same polynucleotide.The character of key or connection may alter a great deal, and connection can zymetology or chemically carry out.Usual zymetology carries out connecting to form phosphodiester bond between 5 ' carbon of the terminal nucleotide at an oligonucleotide and 3 ' carbon of another oligonucleotide.The ligation that many combinations drive describes in the following references:: Whitely etc., U.S. Patent number 4,883,750; Letsinger etc., U.S. Patent number 5,476,930; Fung etc., U.S. Patent number 5,593,826; Kool, U.S. Patent number 5,426,180.
The disclosure provides for removing less desirable material (comprising unconjugated genomic dna and the probe be not connected) and selecting or be separated the method and composition of the material (comprise and connect product) expected.Under in connection, product is the certain situation of ring-type, as and MIP reaction in, utilize Exonucleolytic ferment treatment to remove unconjugated genomic dna and the probe be not connected.In some cases, then utilize the enzyme of the uracil residues depurination in probe, as uracil-N-glycosylase process, the connection product of release ring-type.In these cases, after heating, cut abasic site, produce linearizing connection product.
Can detect in many ways.In some cases, connecting product by droplet digital pcr reaction detection, wherein carrying out synthetic DNA by extending in individual molecule at least one detection probes comprising fluorescent agent-quencher right.Fluorescent agent refers to the molecule (such as, fluorophore) launched after absorb light or other electromagnetic radiation and can detect light.Quencher refers to the molecule reducing material fluorescence intensity, and when fluorescent agent-quencher is right, quencher is by absorbing the detected light that covalently bound fluorescent agent is launched and the detection reduced fluorescent agent.In DNA building-up process, polysaccharase, as 5 ' → 3 ' exonuclease activity cutting detection probes of Taq polysaccharase, causes fluorescent agent to discharge from quencher.Many fluorescence detection methods can detect the fluorescent agent of release, but fluorescent agent-quencher pair can not be detected.In some embodiments, the quantitative assay to the particular sequence (as the target in fetus or mother's genetic material or canonical sequence) existed is provided to the detection connecting product.
The disclosure can be provided for the composition and the method that detect interested nucleic acid molecule further, and wherein sample can comprise DNA, RNA or cDNA from any biology of detecting with droplet digital pcr.In some cases, use ligation sample separation, in some cases, thereafter with Exonucleolytic ferment treatment to remove undesired material.In some embodiments, by detecting the fluorescence monitoring of droplet digital pcr, wherein droplet comprise that PCR reagent in the aqueous phase that is suspended in emulsion and available PCR reaction detection arrive one or more connect products.
The acquisition of tissue and preparation
The method and composition of the disclosure provides the means obtaining fetus or maternal inheritance material.Described method and composition provides when not needing invasive surgical procedures, amniocentesis, Chorionic villi sampling etc., detects the copy number difference of target polynucleotide.In other cases, described method and composition provides the detection of the copy number difference to target polynucleotide in sample (such as blood sample), and it is used as team and has more invasive inspection as the interpolation of surgical procedures, supplementary, preliminary step or auxiliary.Usually, fetus/maternal inheritance material is obtained by blood drawing or other method provided herein.In some preferred embodiments, parent material is that Maternal plasma or peripheral blood are as maternal peripheral venous blood.Can for particular cell types (such as, monocyte; Red corpuscle; CD4+ cell; CD8+ cell; B cell; T cell; NK cell etc.) human peripheral blood cell carries out enrichment.Also alternative particular cell types (such as, the monocyte got rid of in peripheral blood cells; Red corpuscle; CD4+ cell; CD8+ cell; B cell; T cell; NK cell etc.).Parent material also can be the monocyte of derived from bone marrow.Parent material also can comprise the tissue directly extracted from placenta (such as, placenta cells) or umbilical cord (such as, huve cell, TSH in omphalorrhagia, cord blood cell).Parent material also directly can be derived from the fetus of shaping, such as fetal tissue, such as, and fetal fibroblast or hemocyte.Parent material also from baby or children, can comprise neonatal tissue.
In some cases, parent material may obtain from hospital, laboratory, clinical or medical laboratory.In some embodiments, from pregnancy at least 4, individuality (such as, the pregnant woman) collected specimens of 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or 26 weeks.In some embodiments, the individual carrier or have affecting, be genetic diseases by genetic diseases occurs or the risk of transmission genetic diseases, wherein genetic diseases be possible to heritable variation as suddenlyd change, inserting, add, delete, transposition, point mutation, Trinucleotide repeat diseases and/or the relevant any disease of single nucleotide polymorphism (SNP).In other embodiments, the female patient at reproduction age taken from by sample, and in some cases, female patient is pregnancy or unknown Pregnancy status.In other cases, individuality be male patient, the accurate father of the male sex or have specific genetic abnormality risk, be diagnosed as or have the male patient of specific genetic abnormality.In some cases, known male patient is subject to impact or its carrier of genetic diseases or heritable variation, or has the risk of specific genetic abnormality, is diagnosed as or has specific genetic abnormality.In some cases, the state of female patient in genetic diseases or heritable variation may be unknown.In further embodiment, sample takes from children or the adult patients of any known or unknown its genetic sequence copy number variation state.In some cases, known children or adult patients are subject to impact or its carrier of genetic diseases or heritable variation.
An advantage of method and composition provided herein is that they can be supported in the relatively early stage and Maternal plasma of pregnancy fetal nucleic acid (such as, DNA, RNA) total concn lower time detect fetal nucleic acid (such as, DNA, RNA).Foetal DNA concentration in parent material may be at least 0.1% of maternal gene group DNA carrying capacity total in maternal sample, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5% or 25%, be preferably at least 3% of total maternal gene group DNA carrying capacity.In some cases, foetal DNA concentration may lower than about 0.1% of maternal gene group DNA carrying capacity total in maternal sample, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5% or 25%.The type polynucleotide that exist with one amount (H) are comprised (such as at parent material, DNA, RNA) and with the polynucleotide of the type existed lower than the amount (L) of H (such as, DNA, RNA etc.) when, in parent material, the concentration of L may be at least 0.1% of the total concn of H in sample, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5% or 25%, be preferably at least 3% of H.In some cases, L may lower than about 0.1% of the total amount of H in sample, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5% or 25%.
In some cases, in order to obtain the enough nucleic acid for testing, gather the blood volume being at least 1,2,3,4,5,10,20,25,30,35,40,45 or 50mL.This blood volume can provide the STb gene of at least 1,000 genome equivalent (GE).At early pregnancy, STb gene exists with general 1,000GE/mL Maternal plasma, and foetal DNA concentration is about about 3.5% of total plasma dna.But, less blood can be gathered for needing the genescreen of lower significance,statistical, or for foetal DNA enrichment DNA sample.Foetal DNA concentration also may change according to the pregnant age of fetus.In some embodiments, by separating red corpuscle, fetal nucleated red blood especially described below (it is different from seedless adult red corpuscle) and enriches fetal DNA or RNA.In other embodiments, red corpuscle can be removed from maternal blood sample, and genetic material can be obtained from Maternal plasma.
In some embodiments, parent material can be the tissue sample comprising solid tissue, and nonrestrictive example comprises brain, liver, lung, kidney, prostate gland, ovary, spleen, lymphoglandula (comprising tonsilla), Tiroidina, pancreas, heart, skeletal muscle, intestines, larynx, esophagus and stomach.In other embodiments, parent material can be the cell comprising nucleic acid, comprises reticular tissue, muscle tissue, nervous tissue and epithelial cell, and the epithelial cell especially exposed is as skin cells and hair cell.In other embodiments, parent material can be the sample comprising nucleic acid from any biology, can from wherein obtaining genetic material and detecting with droplet digital pcr described herein.
The enrichment of fetal material
Fetal cell can from comprise fetus and mother cell mixture maternal sample enrichment.Fetal concentrations is at least 0.1% of total maternal gene group DNA (or RNA) carrying capacity wherein, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, under the certain situation of 24.5% or 25%, this enrichment may occur.Fetal concentrations exceedes 0.1% of total maternal gene group DNA (or RNA) carrying capacity wherein, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, under the certain situation of 24.5% or 25%, this enrichment may occur.
In some embodiments, by affine method enriches fetal cells, the method can be included in coupling to be had fetal cell than having the molecule of larger avidity as the solid structure of fetal specific antibody collects fetal cell to non-fetal cell.The non-limitative example of solid structure comprises the surface of polymer surfaces, magnetic micro-beads, polymer bead and microfluidic channel.In some embodiments, before method described herein or composition or as its part, not for fetal cell enriched biological sample.In some embodiments, not by affine method enriches fetal cells.In some embodiments, not by using fetal specific antibody enriches fetal cells.In some cases, not by sample is added enriches fetal cells in microfluidic device.
Flow cytometry technique also may be used for enriches fetal cells (Herzenberg etc., PNAS76:1453-1455 (1979); Bianchi etc., PNAS87:3279-3283 (1990); Bruch etc., PrenatalDiagnosis11:787-798 (1991)).U.S. Patent No. 5,432,054 also illustrates the technology using the pipe had bottom wide top and narrow capillaries be made up of polyethylene to carry out isolating fetal erythroblast.In some cases, the fetal cell in the sample not using flow cytometry enrichment to analyze for method of the present invention or composition.The centrifugal density based on molecule of variable velocity program is used to cause red corpuscle stacking in kapillary.Recovery comprises the density portion of low density red corpuscle (comprising fetal erythrocyte), and then differentiation haemolysis is with preferential destruction maternal red blood cells.Ooze the density gradient separation red corpuscle in medium with height, be now rich in fetal erythrocyte relative to lymphocyte and the mother cell broken.Use hypertonic solution to reduce red corpuscle, increase their density, and contribute to carrying out purifying from more highdensity lymphocyte.After isolation of fetal cells, the standard technique purifying foetal DNA of this area described in detail herein can be used.
In some embodiments, maternal blood can be processed with the foetal DNA concentration in enrichment STb gene, as Li etc., described in (2005) J.Amer.Med.Assoc.293:843-849.In brief, commercially column technology (such as, the high-purity template DNA purification kit of Roche can be used; Roche) from 5-10mL Maternal plasma, Circulating DNA is extracted in conjunction with vacuum pump.After extraction, can sepharose (1%) electrophoresis (Invitrogen) DNA isolation be passed through, carefully can cut out the gel section comprising size and be about the Circulating DNA of 300 Nucleotide.Can with extracting test kit (QIAEXIIGelExtractionKit; Qiagen) from this gel film, extract DNA, and be eluted to the aseptic 10-mMTRIS-hydrochloric acid that final volume is 40-μ L, in pH8.0 (Roche).
In some embodiments, from the foetal DNA of separated free the maternal blood sample comprising full cell.In preferred embodiments, the foetal DNA of separated free from Maternal plasma sample.In some embodiments, plasma sample at least 50%, 75% or 95% is not containing fetal cell.In some embodiments, blood plasma is not completely containing fetal cell.
Disclosed in the 15 days July in 2004, " Methodsfordetectionofgeneticdisorders " by name, Dhallan, the U.S. Patent application 20040137470 of RavinderS describes the enrichment procedures of foetal DNA, wherein blood is collected into 9mlEDTAVacuette pipe (article No. NC9897284), in each pipe, add 10% neutral buffer that 0.225ml comprises formaldehyde (4%w/v), each pipe is put upside down gently.Pipe is stored in 4 DEG C until prepare for the treatment of.The reagent of block cell cracking or stabilizing cell membrane can be added in pipe, include but not limited to the derivative of formaldehyde, formaldehyde, formalin, glutaraldehyde, Glutaraldehyde Derivative, linking agent, primary amine reaction linking agent, sulfydryl reactant cross-linker, sulfydryl addition or disulfide reduction, carbohydrate reactant cross-linker, carboxyl-reactive linking agent, photoreactivity linking agent, decomposable asymmetric choice net linking agent etc.The stabilizing cell membrane of any concentration or the reagent of block cell cracking can be added.In preferred embodiments, not hinder or to hinder the concentration of subsequent reactions to add the reagent of stabilizing cell membrane or block cell cracking.
In another embodiment, by the technology and/or the scheme DNA isolation that significantly reduce mother body D NA amount in sample, include but not limited to the Centrifuge A sample when the damping force of whizzer being set to 0 (whizzer does not use braking), when minimally or not disturbance " buffy coat " supernatant liquor is transferred in new pipe, and only a part of supernatant liquor to be transferred in new pipe.In preferred embodiments, the accelerating force of whizzer and damping force are all set to 0.In another embodiment, by the remarkable technology and/or the scheme DNA isolation that reduce the amount of mother body D NA in sample, include but not limited to the Centrifuge A sample when the accelerating force of whizzer being set to 0, when minimally or not disturbance " buffy coat " supernatant liquor is transferred in new pipe, and only a part of supernatant liquor to be transferred in new pipe.In another embodiment, before taking-up supernatant liquor, " buffy coat " is removed from pipe by any applicable method, include but not limited to use syringe or syringe needle to take out " buffy coat ".In another embodiment, the damping force of whizzer is set to certain per-cent of maximum braking force, includes but not limited to 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%.
In another embodiment, the accelerating force of whizzer is set to certain per-cent of maximum accelerating force, includes but not limited to 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%.In another embodiment, the present invention relates to the composition comprising free fetal dna and free mother body D NA, wherein the relation of the free fetal dna that comprises of said composition and free mother body D NA includes but not limited to, is at least about the free fetal dna of 15%, be at least about the free fetal dna of 20%, be at least about the free fetal dna of 30%, be at least about the free fetal dna of 40%, be at least about the free fetal dna of 50%, be at least about the free fetal dna of 60%, be at least about the free fetal dna of 70%, be at least about the free fetal dna of 80%, be at least about the free fetal dna of 90%, be at least about the free fetal dna of 91%, be at least about the free fetal dna of 92%, be at least about the free fetal dna of 93%, be at least about the free fetal dna of 94%, be at least about the free fetal dna of 95%, be at least about the free fetal dna of 96%, be at least about the free fetal dna of 97%, be at least about the free fetal dna of 98%, be at least about the free fetal dna of 99% and be at least about the free fetal dna of 99.5%.
Further, the reagent of stabilizing cell membrane can be added maternal blood to reduce mother cell cracking, include but not limited to aldehydes, urea aldehyde, phenol formaldehyde (PF), DMAE (dimethylaminoethanol), cholesterol, cholesterol derivative, the magnesium of high density, vitamin-E, vitamin e derivative, calcium, calglucon, taurine, nicotinic acid, hydroxylamine derivative, bimoclomol (bimoclomol), sucrose, astaxanthin, glucose, amitriptyline, hopance tetraacethyl phenyl ester (hopanetetralphenylacetate) isomer A, hopance tetraacethyl phenyl ester isomer B, citicoline, inositol, vitamins B, vitamin B complex, cholesterol hemisuccinate, sorbyl alcohol, calcium, ubiquinone, ubiquinone, vitamin K, vitamin K mixture, methyl naphthoquinone, zonisamide, zinc, Semen Ginkgo extrac, Phenytoin Sodium, perftoran, polyvinylpyrrolidone, phosphatidylserine, carbamazepine, PABA, sodium cromoglycate, sodium nedocromil, phenyloin, zinc citrate, mexiletine, Di Lanting, hyaluronate sodium or PLURONICS F87.In another embodiment, the reagent of protection or stabilized cell structural integrity can be used to reduce the amount of lysis.In another embodiment, any scheme of the amount reducing mother body D NA free in maternal blood can be used before obtaining sample.In another embodiment, before acquisition sample, conceived women does not carry out body movement, rest for some time, include but not limited to 0-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-120, 120-180, 180-240, 240-300, 300-360, 360-420, 420-480, 480-540, 540-600, 600-660, 660-720, 720-780, 780-840, 840-900, 900-1200, 1200-1500, 1500-1800, 1800-2100, 2100-2400, 2400-2700, 2700-3000, 3000-3300, 3000-3600, 3600-3900, 3900-4200, 4200-4500 and more than 4500 minutes.In another embodiment, after the female organism of pregnancy reaches relaxation state, sample is obtained from it.Rest period before acquisition sample can reduce the amount of maternal nucleic acids in sample.In another embodiment, obtain sample in the morning (a.m.) from the women of pregnancy, include but not limited to 4-5am, 5-6am, 6-7am, 7-8am, 8-9am, 9-10am, 10-11am and 11-12am.In another embodiment, slept in the women of pregnancy and include but not limited to 0-1,1-2,2-3,3-4,4-5,5-6,6-7,7-8,8-9,9-10,10-11,11-12 or obtain sample from it after for some time more than 12 hours.In another embodiment, before acquisition sample, conceived women sport for some time, subsequently rest for some time again.In another embodiment, 0-15,15-30,30-45,45-60,60-120,120-240 is included but not limited to during motion or more than 240 minutes.In another embodiment, the reagent that will prevent DNA from destroying can be added in blood sample, include but not limited to dnase inhibitor, zinc chloride, ethylenediamine tetraacetic acid (EDTA), Guanidinium hydrochloride, guanidinium isothiocyanate, N-Sarkosyl L and sodium lauryl sulphate.In another embodiment, obtain foetal DNA from fetal cell, wherein said fetal cell is separable from including but not limited to the sources such as the mucous membrane that maternal blood, Cord blood, Chorionic villi, amniotic fluid, embryonic tissue and uterine cervix or vagina from mother obtain.
In another embodiment, the blood collecting technology of any reduction lysis amount, method, step or equipment can be used, include but not limited to the shorter pin of heavy caliber pin, length, increase the pin coating of laminar flow such as Teflon, increase the amendment on the pin inclined-plane of laminar flow or reduce the technology of rate of blood flow.Fetal cell probably in maternal blood by the immune system destruction of parent.But probably most mother cell cracking is due to blood sampling or the process of blood sample.Therefore, to prevent or the method that reduces lysis will reduce the amount of mother body D NA in sample, and increase the relative percentage of free foetal DNA.
Use the scheme of this reagent example as follows: blood storage is in 4 DEG C until process.Pipe to be set in the whizzer of 0 with 1000rpm centrifugal 10 minutes in damping force.Pipe second time centrifugal 10 minutes with 1000rpm.The supernatant liquor (blood plasma) of every increment product is transferred in new pipe, when damping force is set to 0 with 3000rpm centrifugal 10 minutes.Supernatant liquor to be transferred in new pipe and to be stored in-80 DEG C.The buffy coat of about 2 milliliters that comprise mother cell is put into independent pipe and is stored in-80 DEG C.
The extraction of DNA or RNA
Can with technology known in the art isolation of genomic DNA from blood plasma (such as Maternal plasma), as used the QiagenMidi test kit from hemocyte purify DNA.DNA can in 100 μ l distilled water wash-out.Also QiagenMidi test kit DNA isolation from the mother cell be included in buffy coat is used.QIAamp circle nucleic acid test kit also can be used for this object, see, such as http://www.qiagen.com/products/qiaampcirculatingnucleicacidkit. aspx.
The method extracting polynucleotide (such as, DNA) also can comprise use liquid extraction (such as, Trizol, DNAzol) technology.
Such as, initial sample (such as blood or blood plasma) can have the initial volume of 15-30ml, from wherein extracting about 100-200ulDNA or other polynucleotide.Then the 200ulDNA being extracted sample can be transformed the final sample that (or concentrated) is more small volume, such as 5ul, 10ul.In some cases, the volume of initial sample may higher than 2 of final sample volume, 5,10,20,30,40,50,75,100,500,1000,5000,10,000,50,000,100,000,500,000 or 1,000,000 times.Final sample also can be added into the sample for generating in the device of droplet.
The volume of final sample can be 1-20ul.In some embodiments, final sample more than 1,5,10,15,20,25,30,35,40,45,50,75 or 100ul.In some embodiments, final sample is less than 1,5,10,15,20,25,30,35,40,45,50,75 or 100ul.In some embodiments, final sample more than 1,5,10,15,20,25,30,35,40,45,50,75 or 100nl.In some embodiments, final sample is less than 1,5,10,15,20,25,30,35,40,45,50,75 or 100nl.In some embodiments, final sample more than 1,5,10,15,20,25,30,35,40,45,50,75 or 100pl.In some embodiments, final sample is less than 1,5,10,15,20,25,30,35,40,45,50,75 or 100pl.
In some embodiments, DNA can be concentrated by known method, comprises centrifugal and uses various enzyme inhibitors (such as, for DNase).DNA can be incorporated into selective membrane (such as silicon-dioxide) and by itself and separated from contaminants.Also the fragment enrichment DNA of 1000,500,400,300,200 or 100 base pairs can be less than for the length circulated in blood plasma.This size is selected to carry out on DNA size separation medium, as running gel or chromatographic material (Huber etc. (1993) NucleicAcidsRes.21:1061-6), gel filtration chromatography, tsk gel (Kato etc. (1984) J.Biochem, 95:83-86).In some cases, polynucleotide (such as, DNA, RNA) or may use solid-phase media selectivity to catch by selective precipitation, concentrated (such as, sample can experience evaporation).After precipitation, DNA or other polynucleotide can be reconstructed or be dissolved in small volume.Small volume can support the hybridization of probe and target polynucleotide, or improves the hybridization of probe and target polynucleotide.
In some embodiments, parent material can comprise cell or tissue, comprises reticular tissue, muscle tissue, nervous tissue, hemocyte or epithelial cell.In some cases, with ferment treatment (as protease digestion), non-nucleic acid substances can be removed from parent material.In some cases, can by with destroying the stain remover of film and/or cleavage method (such as, supersound process, Fu Shi crush method (Frenchpress), freeze/thaw, dounce homogenate) process and remove other non-nucleic acid substances, subsequently can be centrifugal the part comprising nucleic acid is separated with the part not containing nucleic acid.This area is adopted to improve the scheme of foundation, the nucleic acid extracted can from any suitable sample, include but not limited to the tissue sample comprising nucleic acid, body fluid (such as, blood, serum, blood plasma, saliva, urine, tears, peritoneal fluid, ascites, vaginal secretions, breast fluid, milk, lymph liquid, cerebrospinal fluid or mucosal secretions), Cord blood, Chorionic villi, amniotic fluid, embryo, the embryo of two cells, the embryo of four cells, the embryo of eight cells, the embryo of 16 cells, the embryo of 32 cells, the embryo of 64 cells, the embryo of 128 cells, the embryo of 256 cells, the embryo of 512 cells, the embryo of 1024 cells, embryonic tissue, lymph liquid, cerebrospinal fluid, mucosal secretions or other body exudates, movement, individual cells, or comprise they nucleic acid these source extracts, with subcellular structure as plastosome.
In preferred embodiments, can to collect blood in the device comprising the magnesium chelating substances including but not limited to EDTA and to be stored in 4 DEG C.Optionally, can calcium sequestrant be added, include but not limited to EGTA.In another embodiment, cell lysis inhibitor can be added to maternal blood, include but not limited to formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, Glutaraldehyde Derivative, protein cross agent, nucleic acid crosslinking agent, protein and nucleic acid crosslinking agent, primary amine reaction linking agent, sulfydryl reactant cross-linker, sulfydryl addition or disulfide bond reduction, carbohydrate reactant cross-linker, carboxyl-reactive linking agent, photoreactivity linking agent, cleavable linking agent.
Blood plasma RNA is extracted in Enders etc. and (2003), describe in ClinicalChemistry49:727-731.In brief, the blood plasma gathered in the crops after centrifugation step can mix with TrizolLS reagent (Invitrogen) and chloroform.Can centrifugal mixture, and water layer is transferred in new pipe.Ethanol is added to water layer.Then according to manufacturer recommends mixture be added on RNeasy micro-column (Qiagen) and process.
In some cases, when extract material comprise single stranded RNA, double-stranded RNA or DNA-RNA heterocomplex time, available technology known in the art by these Molecular Cloning be double-stranded DNA.Such as, reversed transcriptive enzyme may be adopted from RNA molecule synthetic DNA.In some cases, RNA may need Connection Step in advance so that linker fragment is connected to RNA to the conversion of DNA, thus allows to use the initial reverse transcription of universal primer.In other cases, the poly A tract of mRNA molecule such as can be used to carry out initial reverse transcription.In some cases, after being converted into DNA, method described in detail herein may be used to catch, select, mark or be separated the sequence of expectation further.
Although this specification sheets mentions foetal DNA all the time, the fetal rna (and common RNA) found in maternal blood also can be analyzed.As previously described, " mRNAofplacentaloriginisreadilydetectableinmaternalplasma; " (Ng etc. (2003) Proc.Nat.Acad.Sci.100:4748-4753), when analyzing with respective real-time PCR assay, in Maternal plasma, detect hPL (human placental prolactin) and hCG (human chorionic gonadotrophin) mRNA transcript.In the method, can be used in placenta and express and the mRNA encoding gene be present on interested karyomit(e).Such as, DSCR4 (territory, Down Syndrome Critical 4) finds and mainly expresses in placenta on karyomit(e) 21.Its mRNA sequence is found in GenBankNM_005867.In this case, RNaseHminus (RNase is preferably used
h-) the next cDNA for the preparation of detecting of reversed transcriptive enzyme (RT).RNase
h-rT can obtain from several manufacturer, as SuperScript (TM) II (Invitrogen).Reverse transcriptase PCR can as used for as described in chromosomal DNA herein.RNA can comprise the RNA of siRNA, miRNA, cRNA, tRNA, rRNA, mRNA or other type any.
Linking probe
In some preferred embodiments, by using one or more bar linking probe (being also sometimes referred to as " attachable probe " at this) to mark, select, catch, be separated and/or process target polynucleotide.Linking probe comprises: (1) probe of cyclisation " can ", wherein each end (5 ' and 3 ') of single polynucleotide (or oligonucleotide) is in conjunction with the close of target polynucleotide or adjacent area, wherein after this combination, 5 ' of probe end can be connected with 3 ' end by ligation, thus this probe of cyclisation; Or (2) two polynucleotide (or oligonucleotide) probe, wherein after two probes are bonded to the region in target polynucleotide, 5 ' end of a probe can be connected with 3 ' end of another probe.After the adjacent or similar sequences of two such probes and target polynucleotide is hybridized, ligation causes two probes to link together becomes a linear probe.
In some embodiments, linking probe also can comprise: restriction enzyme site, universal primer site and/or general probe binding site.In some embodiments, linking probe is phosphorylated at its 5 ' end.In other embodiments, linking probe is not phosphorylated at its 5 ' end.3 ' the end of same (or different) linking probe that the phosphorylation of 5 ' end like this can allow this 5 ' end to be connected to combine from the adjacent area of target polynucleotide, and do not need gap fill to react.In other cases, synthesize at the unphosphorylated probe of 5 ' end.In this case, designing probe and make the calmodulin binding domain CaM of 5 ' end contiguous but the binding site of the direct 3 ' end of adjacent same (or different) probe.Connect this probe and may also need gap fill or extension.
In some embodiments, linking probe is molecular inversion probes.United States Patent (USP) 7,368,242 describe molecular inversion probes and how it is used for producing amplicon after interacting with the target polynucleotide in sample.Under the adjacent area allowed in hereditary target and its complementary region in molecular inversion probes (or other linking probe) form the condition of stable duplex, the linear forms of probe are combined with the sample comprising target polynucleotide.Usually, one of 5 ' end of probe and target sequence combine, and 3 ' end of probe is combined with adjacent sequence, thus form ring structure.End possibility (incision separation) adjacent to each other in desired specificities region, or the breach of several Nucleotide (such as 1-10 Nucleotide) between them, may be had.In some embodiments, breach is more than 5,10,20,30,40,50,100,200,300,400 or 500 Nucleotide.In some preferred embodiments, target specificity region direct neighbor (such as, 0, interval Nucleotide).No matter which kind of situation, after target specificity area hybridization, the end in two target specificity regions is covalently bound in the following manner: the ligation utilizing gap fill to react or the multiple extension utilizing gap fill to react and ligation subsequently.
Fig. 4 uses molecular inversion probes (MIP) to detect two kinds of genetic targets target diagrams.A hereditary target is identified by a probe (MIP1-1), and second hereditary target identify by Article 2 probe (MIP2-1).After MIP is bonded to hereditary target, the 5 ' end carrying out the combining MIP probe of ligation is connected to 3 ' end, thus forms ring-type MIP.In some cases, MIP probe combines the adjacent DNA sequence dna of two of being separated by one or two Nucleotide.In this case, gap fill (or extend) reaction is carried out to use target DNA to carry out fill up the gap for template.After MIP is in conjunction with its target sequence, MIP forms ring, and the sequence of probe may be inverted (see Fig. 4).Can carry out ligation after this inversion, the end being wherein inverted molecule couples together the probe forming cyclisation.
After MIP probe is combined (and optionally gap fill reacts) with DNA, carry out ligation with cyclisation MIP probe with ligase enzyme.Then in exonuclease digestion process, the MIP probe of ring-type is retained, the untapped linear single-stranded probes of this exonuclease digestion, the linear genomic dna of strand and double stranded genomic dna.Then ring-type MIP and PCR reagent are combined in droplet, to be analyzed by droplet digital pcr.In some embodiments, PCR reaction before or period by cycling probe linearizing.As shown in Figure 4, probe can comprise the site containing restriction enzyme site (such as, cutting responsive a series of uracil residues to uracil-N-glycosylase enzyme).In some cases, there is enzyme and cut step, wherein polynucleotide are cut and form linear molecule.In other situation, in described step, do not have enzyme to cut step, polynucleotide maintain ring-type state.Next, the MIP probe of connection (or ring-type, or linearly, or both mixtures) subdivided in one of multiple subregion.Preferably, this subregion is droplet (the water-based droplet such as, in oil phase).Then droplet carries out thermal cycle reaction.During thermal cycle reaction, linearizing MIP (or, in some cases, the MIP of ring-type) as reaction template by general forward primer (UF1 or UF2) and general reverse primer (UR1 or UR2) initial amplification.During increasing, cutting and the general probe (UP1 or UP2) of the sequence hybridization in every bar MIP, be separated with the quencher side of the fluorescence side and probe that make probe.As the result of this cutting, the fluorescence from the fluorescent agent side of probe increases.
In some embodiments, the polysaccharase by having 5 '-> 3 ' polymerization activity carries out gap fill reaction.The polysaccharase that can be used in this method is included in those polysaccharases of initial 5 '-3 ' polymerization in nicking sites place.The chain of the polysaccharase also polymerization in removable otch downstream.In some embodiments, the polysaccharase for gap fill reaction lacks any 5 '-> 3 ' exonuclease activity.In a case where, the polysaccharase usually with this exonuclease activity can lack this activity: add encapsulant and block this activity; Exercise structural domain or fragment deletion, sudden change or the modification of the polysaccharase of 5 '-> 3 ' exonuclease activity; Polysaccharase is modified by sulphation; Or any other method known in the art.
In some embodiments, the polysaccharase for gap fill reaction comprises 3 '-> 5 ' editor exonuclease activity.The example of suitable polysaccharase comprises the klenow fragment of DNA polymerase i, the klenow fragment of DNA polymerase i of exonuclease disappearance and the similar fragments (Bio-Rad, Richmond, Calif.) from Bst polysaccharase.SEQUENASE1.0, SEQUENASE2.0 (USBiochemical), T5DNA polysaccharase and Phi29DNA polysaccharase also work, (LifeTechnologies as the Stoffel fragment of AmpliTaqDNA polysaccharase, Carlsbad, CA).
Although the disclosure describe comprise DNA linking probe (such as, MIP probe), linking probe described herein can comprise other nucleic acid (such as, RNA, mRNA, cDNA, rRNA, tRNA, siRNA, miRNA etc.), polypeptide, the nucleic acid of synthesis or polypeptide of synthesis any.In some cases, linking probe can comprise two or more dissimilar polynucleotide (such as, not only comprising RNA but also comprise DNA), or linking probe can comprise polynucleotide and polypeptide (such as, RNA+ polypeptide; DNA+ polypeptide).In other application specific, in method described herein, linking probe (such as, MIP probe) can be coupled to fluorescence dye, solid support or microballon.
Nucleic acid refers to the nucleic acid that naturally occurring and non-natural exists, and with the nucleic acid analog that the mode similar to naturally occurring nucleic acid works.Nucleic acid can be selected from RNA, DNA or nucleic acid analogue molecule, as sugar or the ribonucleotide of backbone modification or deoxyribonucleotide.Other nucleic acid analog such as peptide nucleic acid(PNA) (PNA) or locked nucleic acid (LNA) are also applicable.The example of the nucleic acid that non-natural exists comprises: the base of halogen substiuted, the base that alkyl replaces, the base that hydroxyl replaces, the base that sulfydryl replaces and 5-propinyl-uracil, 2-sulfo--5-propinyl-uracil, 5-methylcytosine, isoguanine, iso-cytosine, false iso-cytosine, 4-thiouracil, 2-thiouracil and 2-sulphur thymus pyrimidine, inosine, 2-aminopurine, N9-(2-amido-6-chloropurine), N9-(2, 6-diaminopurine), xanthoglobulin, N9-(7-denitrogenation-guanine), N9 (7-denitrogenation-guanozola), N8 (7-denitrogenation-guanozola), 2-amino-6-" h "-purine, 6-amino-2-" h "-purine, 6-oxo-2-" h "-purine, 2-oxo-4-" h "-pyrimidine, 2-oxo-6-" h "-purine, 4-oxo-2-" h "-pyrimidine.Those form the base pair of two hydrogen bonds by with non-sulfhydrylation and sulfhydrylation base; Be respectively 2,4-dioxo and 4-oxo-2-thiopyrimidine, 2,4-dioxo and 2-oxo-4-thiopyrimidine, 4-amino-2-oxo and 4-amino-2-thiopyrimidine, 6-oxo-2-amino and 6-sulfo--2-aminopurine, 2-amino-4-oxo and 2-amino-4-thiopyrimidine, 6-oxo-2-amino and 6-sulfo--2-aminopurine.
In some preferred embodiments, present method comprises the sequence selecting, mark, catch and/or be separated expectation by optionally protecting the sequence of expectation to avoid enzymic digestion (as endonuclease and the enzyme of exonuclease) from genomic dna.Such as, the cyclisation (after it is in conjunction with its target) of MIP probe protects probe from the digestion of some enzyme (such as, exoI, exoIII).Also can use other after probe is in conjunction with its target, protect the method for probe.
In some cases, it can be enzymic digestion after ligation, as Exonucleolytic ferment treatment (such as, exonuclease I, exonuclease III), to digest unconjugated genomic dna and unconjugated probe, but indigestion cyclic DNA, thus the ring-type MIP being separated that sequence is expected in representative.In some cases, when combining the hereditary target expected more than 1 probe and when carrying out connecting to form ring-type MIP, MIP allows multiplex.So multiple MIP can represent given hereditary target, enhances the sensitivity of detection.
Generation ring-type MIP with under the certain situation representing interested sequence, can before PCR reaction detection (or period) by these ring-type MIP linearizings.In some cases, MIP comprise available enzyme as uracil-N-glycosylase ferment treatment the uridylic base of depurination, and ring molecule can become linearizing at abasic site after the heating.In other cases, MIP can comprise locus specificity Restriction Enzyme for restriction enzyme site, cutting cycling probe to form linear DNA molecule.In some embodiments that the MIP of ring-type is linearized wherein, before MIP linearizing, by methods such as such as hot deactivation, pH sex change or physical sepn by the enzyme comprised in the solution of MIP (comprising exonuclease) deactivation.In some cases, gel available purifying or alcohol settling, or can by organic solution as trichloroacetic acid precipitation remove protein from solution from protein separation DNA.
The example of Restriction Enzyme comprises AatII, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpul0I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsCI, BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DraIII, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, KasI, KpnI, MboI, MboII, MfeI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I, MspI, MwoI, NaeI, NarI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PciI, PflFI, PflMI, PhoI, PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, StyD4I, StyI, SwaI, T, Taq α I, TfiI, TliI, TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI and ZraI.
The probe of other type and the method for other screening-gene probe also can be used in method and composition described herein.Such as, although the use of MIP probe is usually directed to the cyclisation of single connection probe, cyclisation step is always unnecessary.Such as, can use joint detection round pcr, wherein two different probes (wherein every bar is hybridized with adjacent DNA (or contiguous DNA)) link together, and add the fragment that universal primer is connected with detection with probe subsequently.
The PCR of Fig. 7 display in joint detection reaction (LDR) and droplet subsequently, with two kinds of color detection, two kinds of hereditary targets.Article two, linear oligonucleotide is incorporated into genetic targets and puts on adjacent or contiguous region.These regions can direct neighbor or separated by breach.In addition, region can be separated by breach, and this breach can fill with polymeric enzyme reaction, and this reaction extends the length of 3 ' end of the first probe and makes its 3 ' end directly adjacent with 5 ' end of the second probe.Then two probes are connected with each other (as shown in Figure 7).During connecting, two linear oligonucleotide couple together and form single template oligonucleotide (LDR1-1 or LDR2-1).This single template oligonucleotide, instead of form the oligonucleotide pair of single template oligonucleotide, product can be produced in the PCR reaction using general forward (UF1 or UF2) and general oppositely (UR1 or UR2) primer.In addition, this PCR reaction comprises general probe (UP1 or UP2), and this probe comprises the fluorescent agent-quencher pair of hybridizing with a part for this template oligonucleotide.Between the PCR reaction period, 5 ' of archaeal dna polymerase (as Taq)--> 3 ' exonuclease activity cutting probe, causes the fluorescent agent end of molecule to depart from its quencher end.As the result be separated between this fluorescent agent probe and quencher probe, in reaction, fluorescence intensity will strengthen, and can detect in the following steps.Two general probes (UP1 and UP2) comprising two kinds of different colours fluorescent agents can be used to carry out this analysis, this two kinds of different colours can be distinguished between detection period.Such as, LDR1-1 can identify that target sequence is as suspicious dysploidy karyomit(e), and LDR2-1 identifies the diploid karyomit(e) of canonical sequence as supposition, thus allows the detection to dysploidy.
The linking probe used in joint detection reaction described herein, once be connected to target polynucleotide, just protectedly can avoid Exonucleolytic ferment treatment.Such as; add blocking group, chemical closed cell or thiophosphoric acid to modify certain exonuclease that the linking probe of hybridization can be protected to avoid by unconjugated probe and/or unconjugated target polynucleotide (such as, genomic dna) can be digested and digested.Thiophosphoric acid is modified and the probe of connection can be protected to avoid the process of the activity of 3 ' to 5 ' exonuclease exoIII.Similarly, thiophosphoric acid is modified and the probe of connection can be protected to avoid the process of the activity of 5 ' to 3 ' exonuclease exoT7.In some cases, 3 ' to 5 ' exonuclease exoT and 5 ' can be used to 3 ' exonuclease RecJf.There is provided open thiophosphoric acid for the protection of exoT activity at Putney etc. (1981) PNAS78 (12): 7350-54.For RecJf, also see Tosch etc., (2007) J.ofPhysics:ConferenceSeries61 (2007) 1241-1245; Doi:10.1088/1742-6596/61/1/245, international nanometer science and technology meeting (ICN & T2006).ExoT and RecJF digests ssDNA, and block by thiophosphoric acid.Thiophosphoric acid is modified can be arranged in the end of the universal PC R primer sequence of probe or the upstream afterbody of universal PC R primer sequence.
In some embodiments, probe comprises the mixture of different linear oligonucleotide, wherein after every bar probe and target polynucleotide are hybridized, 5 ' region of a linear oligonucleotide can with 3 ' joint area of another linear oligonucleotide.In some embodiments, article two, identical (or substantially identical) oligonucleotide can be incorporated into the close of target polynucleotide or adjacent area in one way separately, and this mode makes 3 ' end of the probe that 5 ' end of such probe then can be such with another be connected.Such connection is all there is in every bar probe and target polynucleotide after hybridizing.
In other embodiments, described method comprises the sequence of catching expectation, and is not separated subsequently.In some cases, the sequence that the linear probe identification more than one is expected also combines with it.Probe can carry out ligation and be interconnected to make one or more probe after combining.In some cases, catch the sequence of expectation due to connection, this can allow to detect by PCR the probe (being called as Ligase detection reaction-PCR or LDR-PCR) connected in a subsequent step, and the probe do not connected cannot be detected by PCR.In some cases, multiple probe can connect in conjunction with hereditary target, which enhances and detects the sensitivity of genetic targets target by LDR-PCR.
Can design linking probe (such as, MIP probe) makes the test between sample and sample show minimize variability or optimize this test in other respects to meet certain standard.Some standards used in the design of linking probe comprise: (1) target sequence does not comprise any known SNP (all SNP such as, in dbSNP); 2) target sequence is positioned at the conservative region target sequence of genomic dna; (3) target sequence is not overlapping with any known CNV region (all CNV existed in the CNV trace such as, in UCSC genome database); 4) in the region of the target polynucleotide (such as, genomic dna, RNA) of target sequence conservative between being positioned at kind (such as, by the conservative follow-up evaluation in UCSC genome database).In addition, in order to optimize the general of probe and consistent performance, some standards can be applied to selecting of target sequence.Can target sequence be selected and make them be unique in human genome.Can target sequence be selected and make two of MIP probe ends comprise G/C Nucleotide thus make their length be approximately 40 Nucleotide, thus make the guid arm of combination (homerarm) have similar melting temperature(Tm) (such as, use the default parameters of Primer3 software, within 67 degree ± 2 degree), and make single guid arm have similar melting temperature(Tm) (such as, use the default parameters of Primer3 software, within 50 degree ± 2 degree).(with 5 ' of the probe of genomic dna complementation and 3 ' end be called as guid arm respectively: H2 and H1.)
Also can screen MIP and target, remove the MIP and target that form secondary structure, because they possibly cannot be incorporated into their counterpart well.In addition, MIP can compare mutually to reduce the possibility of reacting between MIP probe in solution.Some are found in (2010) such as Hyman for avoiding the generic principles of secondary structure, AppliedandEnvironmentalMicrobiology76:3904-3910.Can by set up dG score distribution and remove outlier help screen secondary structure.
Method provided herein comprise for while evaluation Example as the multiple abnormal method on karyomit(e) 13,18 and 21.For this research, karyomit(e) can be used as reference each other, therefore, may not need other with reference to sample or with reference to probe (such as karyomit(e) 1).
Comprise the genomic dna that genetic targets target sample can comprise complete chromosome, chromosome segment or non-chromosomal form.In some cases, the mean length of genomic DNA fragment can be less than about 100,200,300,400,500 or 800 base pairs, or be less than about 1,2,5,10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190 or 200 Nucleotide, or be less than about 1,2,5,10,20,30,40,50,60,70,80,90,100 kilobase.In some cases, the length range of fragment is 10-500,10-1000 or 100-150 base (or Nucleotide), in some embodiments, and a preferred 100-150 base.
Under the certain situation of Fetal genome DNA compared to mother body D NA enrichment, clip size on average may be about 300 base pairs or 100 or 150 base pairs.In some cases, sample comprises at least one genome equivalent.In other cases, sample comprises and is less than a genome equivalent, but comprises enough genomic dnas in order to determine the ratio of target and canonical sequence in fetus or maternal sample.In other cases, sample comprises about half genome equivalent.The term " genome equivalent " used refers to the distribution based on the Genome Size of calculating and the sample DNA of DNA Weight computation, wherein haploid genome heavily about 3.3pg, the genomic content heavily about 6.6pg of diploid normal cell (46 karyomit(e)), corresponding to two genome equivalents (GE) (" genomic equivalent " and " genome equivalent " are used interchangeably herein).In practice, DNA sample size may have some to change.In addition due to random fragment distribution, given genome equivalent may not comprise the DNA fragmentation just only corresponding to a complete diploid gene group.
Multiplex
Described herein and amplification method known in the art (such as, PCR) by multiplex, namely can run with multiple primer and probe in each reaction volume.In the embodiment of some method and compositions provided herein, in given sample volume, have at least 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,100,200,500,1000,5000,10,000,20,000,30,000,40,000,50,000,60,000,70,000,100,000,2,000,000,3,000,000,4,000,000,5,000,000,6,000,000,7,000,000,8,000,000,9,000, or more article 000 or 10,000,000, different probe.In some embodiments, in given sample volume, have at least 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,100,200,500,1000,5000,10,000,20,000,30,000,40,000,50,000,60,000,70,000,100,000,2,000,000,3,000,000,4,000,000,5,000,000,6,000,000,7,000,000,8,000,000,9,000, or more article 000 or 10,000,000, primer.
In some embodiments, use multiple probe (or primer sets), and these probes (or primer sets) are different in one or more.Probe can in conjunction with identical target polynucleotide; Or different target polynucleotide (such as, different karyomit(e); Or identical karyomit(e), but described intrachromosomal different zones).Such as, can use for different target more than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 kind of probe.In some cases, can use for different target more than 20,30,40,50,100,500,1000,5000,10000,50000,100000,500000,1000000 kind or more plant probe.In other cases, there is dissimilar cleavage site in probe.In some cases, described multiple probe comprises many dissimilar probes (such as, linking probe, MIP, snap close probe, PCR primer group, universal primer, general probe and arbitrary combination thereof).In some cases, the difference of described many dissimilar probes is that every bar probe conjugate is in different signal reagent (such as, green fluorescence group, red fluorescence group, etc.).In some cases, the difference of probe is that they comprise different primer binding sites.In other cases, the universal primer of identical group can be used in conjunction with all or most of probe in described multiple probe.
At reaction volume as the multiplex in droplet allows to detect the minor alteration of the DNA ratio between target and canonical sequence relative to 1: 1 ratio desired by diploid sequence.Multiplex allows to count a large amount of sequences, although GE/mL is very low in sample (such as, 1000GE/mL), as in Maternal plasma for the target organized arbitrarily and canonical sequence.Complete target and reference karyomit(e) are macromole, and have and can be guarded and the region of uniqueness by the identification of Auele Specific Primer group and the multiple of amplification.In blood plasma, Circulating DNA can be used as small segment and there is (about 300bp).The multiplex primer of little product (such as, 100 base pairs) is produced, the small segment (such as, 300 base pairs) of can effectively increase target or canonical sequence by design.
Multiplex can increase the possibility being separated in reaction volume and being identified by one of multiplex primer as the target in droplet.Multiplex also can increase the possibility that amplification occurs, and the target sequence being counted as feminine gender in substance test can be made to be measured as the positive.Like this equally for canonical sequence.In some embodiments, the degree of multiplex can comprise for target sequence more than a primer sets, as at least 2,3,4,5,10,15,20 or 25 primer sets, each for specific target sequence.In some embodiments, the degree of multiplex can comprise the primer sets of the reference more than for canonical sequence, as at least 2,3,4,5,10,15,20 or 25 primer sets, each for specific canonical sequence.In some embodiments, the degree of multiplex can comprise for target sequence more than a primer sets and for canonical sequence more than one with reference to primer sets, as at least 2,3,4,5,10,15,20 or 25 primer sets for specific target or canonical sequence.In some embodiments, not identical with the number of the primer sets for canonical sequence for the number of the primer sets of target sequence.In some embodiments, the degree of multiplex can be less than 500,250,200,150 or 100 primer sets for each target and canonical sequence.Target and canonical sequence multiplex can be combined in a reaction volume.
The sequence of different primers to amplification can be distinguished based on spectrally differentiable probe (the such as probe of 2 kinds of different dyes marks, as Taqman or hold lock nucleic acid probe (UniversalProbeLibrary, Roche)).In this approach, the difference in all probe combinations a to reaction volume and based on the color of every bar probe emission is distinguished.Such as, for a kind of polynucleotide (such as, test chromosome, karyomit(e) 21) probe can with the dye-coupling with the first color, and in reaction for the second polynucleotide (such as, with reference to karyomit(e), karyomit(e) 1) probe can with the dye-coupling with the second color.Then the ratio of color reflect test with reference to interchromosomal ratio.
In some cases, one group of probe (such as, one group of probe for test chromosome such as karyomit(e) 21) can for the different zones of target polynucleotide, and the every bar probe in this group has identical universal primer binding site.In some cases, every bar probe has identical probe binding site.In some cases, in reaction, the probe of two or more can have different probe binding site.In some cases, probe conjugate in this reaction is added in identical signal reagent (fluorophore of such as same color).In some cases, different signal reagents (such as, two kinds of distinct colors) is coupled to one or more bar probe.
Alternatively, reaction volume (such as droplet) group can be divided into two sample sets, one group of amplification target sequence, another group amplification canonical sequence.Then target and reference gene is measured independently of one another.This allows to use a kind of fluorescent probe as SYBRGreen.In some cases, this needs sample be divided into two parts and doubled to obtain equal sensitivity by the primer number in each multiple group possibly.In some cases, sample is divided into two parts, the multiple linking probe for test chromosome is added in the half of sample, and add for reference to chromosomal multiple linking probe in second half of sample.In the present case, then linking probe can hybridize with the general probe being coupled to identical signal reagent (such as the fluorophore of same color spectrum).
Also the probe for target can be used in early days in step, instead of with primer pair, to complete the multiplex that the disclosure provides.Operable exemplary probe has the linear oligonucleotide be designed to especially with two ends of adjacent or contiguous sequence hybridization in target polynucleotide.The non-limiting example of this probe is snap close probe, and it is that linear oligonucleotide has two ends being designed to especially hybridize with adjacent target sequence.Once hybridization, two ends can combine by connecting, and snap close probe becomes cyclisation.Snap close probe is at (1998) NatGenetics19:225-232 such as such as Lizardi; United States Patent (USP) 5,871,921; 6,235,472; With 5,866, open in 337.In some cases, probe (such as oligonucleotide) is incorporated into the flanking sequence of genomic dna, and then end directly connects by ligase enzyme reaction.In other cases, between two ends, there is the breach of one or more base.In this case, can carry out extending or gap fill reaction.For gap fill reaction, any method known in the art can realize.Such as, can the mixture of Nucleotide (dATP, dCTP, dGTP, dTTP, dUTP) and polysaccharase, ligase enzyme and other reacted constituent be joined in reaction mixture, hatch about 10 minutes at about 60 DEG C, hatch about 1 minute at 37 DEG C subsequently.Be combined with target polynucleotide and after connecting, linking probe (such as, molecular inversion probes, snap close probe etc.) can be changed into cyclisation.
In some cases, probe is described herein in conjunction with genetic targets target oligonucleotide probe.In some cases, probe is in conjunction with reference target target oligonucleotide probe.Reference target target example is karyomit(e) 1 or other unlikely relevant with fetus dysploidy karyomit(e).In some cases, oligonucleotide or reference oligonucleotide comprise the site that can digestedly cut.Such as, oligonucleotide can be comprise a series of one or more uracil residues, the DNA oligonucleotide of such as at least 1,2,3,4,5,6,7,10,15 or 20 uracil residues, and can by enzyme as uracil-N-glycosylase (UNG) cutting.In other cases, oligonucleotide can comprise one or more restriction site.Oligonucleotide can comprise one or more identical restriction site or one or more different restriction site.The example of restriction site is known in document.Usually, the site that being limited property enzyme can be used to cut.Restriction Enzyme can be can at any Restriction Enzyme (or endonuclease) of specific site cutting.In some cases, Restriction Enzyme is flush end nickase; In other cases, Restriction Enzyme cuts in asymmetric site to produce overhang.There is provided herein the non-limiting example of Restriction Enzyme.
Oligonucleotide probe can comprise the site of hybridizing with forward and reverse primer such as universal primer further.Universal primer used herein comprises and identifies and hybridize to a pair of sequence of both sides, region to be amplified or more 5 ' and 3 ' primer.Region to be amplified can be arranged in the karyomit(e) of hereditary target as fetus dysploidy under a cloud, and so chromosomal non-limiting example comprises karyomit(e) 21, karyomit(e) 13, karyomit(e) 18 and X chromosome.In some cases, region to be amplified is in the chromosomal hereditary target of diploid of supposition.
In some cases, region to be amplified not in hereditary target, and for genetic targets target probe as in molecular inversion probes.Primer pair can for hereditary target, or they can be the sequences of universal primer identification numerous amplification target flank.Such as, can be comprised one or more for genetic targets target probe and identify and the section being incorporated into particular sequence in hereditary target, and probe can comprise the total universal sequence of one group of all probe extraly.Therefore, a pair universal primer can be used for any probe of increasing in this group.In some cases, universal primer is to only just producing detectable PCR primer when molecular inversion probes is inverted.Can by the site in cutting ring-type molecular inversion probes, the inversion of inducing molecule inversion probes, this cutting result in the inversion of primer relative to the direction of its primer pair.In some cases, universal primer, to only when increasing the product of ligation, such as in joint detection reaction, just produces detectable PCR primer.
Oligonucleotide probe also can comprise the sequence as complementary in the probe (such as, TaqMan probe) of dyestuff or fluorescence dye with being connected to marker.In some cases, TaqMan probe is incorporated into the dyestuff (such as, FAM, VIC, TAMRA, ROX) of a type.In other cases, oligonucleotide has more than a TaqMan probe site, each site can in conjunction with different TaqMan probe (such as, having the TaqMan probe of dissimilar dyestuff).Also may have multiple TaqMan probe site, they have the sequence identical with oligonucleotide probe described herein.Usually, TaqMan probe only may be incorporated into the site on oligonucleotide probe described herein, and is not incorporated into genomic dna, but in some cases, TaqMan probe may in conjunction with genomic dna.
Use the advantage of oligonucleotide described herein to be, compare and use Standard PCR technology, as used the technology of primer sets, signal improves more than 1,2,5,10,15,20,30,40,50,75 or 100 times with background noise ratio.A reason is, for for particular target as chromosomal all oligonucleotide probes may only need a kind of probe.Such as, a large amount of oligonucleotide probes may be had (such as, more than 50 kinds), wherein often kind is incorporated into sites different on karyomit(e), but wherein often kind also comprises general or identical TaqMan site, therefore when the TaqMan probe being bonded to specific fluorescent dyestuff fluoresces at identical wavelength with during probe anneals.
Method provided herein comprises the method with following steps: the sex change and the annealing steps that allow one or more oligonucleotide probe and genomic DNA hybridization.Optional gap fill reaction (if 5 ' of probe and 3 ' end not directly for the flanking sequence of genomic dna), the ligase enzyme of following by circularizing probes reacts.The method can comprise exonuclease treatment step further, wherein with exonuclease sample as described in exonuclease I and/or III process, this enzymic digestion linear probe (namely, there is no the probe of successful cross) and ssDNA and dsDNA is (such as, genomic dna), be inverted step subsequently.The method can comprise amplification step further, wherein adds in sample PCR reagent such as Taq polysaccharase, universal primer, fluorescent probe (such as TaqMan probe) and other PCR reactive component with one or more sites of increasing on oligonucleotide probe.The method can comprise allocation step further, be wherein monodispersed water-in-oil droplet by sample emulsification, such as, more than 1,000,10,000,20,000,50,000,100,000,200,000,500, the droplet (being also referred to as reaction volume herein) of 000 or more, follows by thermal cycling, and detects the fluorescence of each droplet at the wavelength place corresponding to fluorescent probe used.In some cases, there is the DNA of average about 1,2,3,4 or 5 copy in each droplet.In some cases, there is average about 0.001,0.005,0.01,0.05,0.1,0.5,1,2,3,4 or 5 oligonucleotide probe in each droplet.Method described herein can be carried out when the height multiplex degree of depth.When the height multiplex degree of depth and ddPCR count in conjunction with time, it can provide a large amount of target counting with the high resolving power realizing relative karyomit(e) dosage.This multiple method quantitatively can combine to prevent false negative with using the ddPCR fetus carrying capacity of the SNP of paternal inheritance, Y chromosome target or fetus specific methylation marker.Fetus carrying capacity mensuration can be carried out separately to the sample that portion extracts, or after the inversion step of test, fetus carrying capacity mensuration can be carried out by the multiplex of two kinds of orthogonal tests (general MIPPCR+ fetus quantitative determination of specificity).
In some cases, connect with general PCR method coupling to realize multiplex.Example includes but not limited to: molecular inversion probes (MIP) strategy (see Hardenbol etc., (2003) NatureBiotechnology, 21 (6): 673-78); U.S. Patent Application Publication No. 2004/0101835; The probe amplification (MLPA) (see SchoutenJP, McElgunnCJ, WaaijerR, ZwijnenburgD, DiepvensF, PalsG (2002), NucleicAcidsRes.30 (12)) of multiple join dependency; Joint detection reaction (LDR); And ligase chain reaction.The general introduction accompanying drawings providing the different multiplexing strategy using dissimilar probe or probe/combination of primers of this specification sheets.
Fig. 5 shows MIP method multiplex to strengthen the sensitivity of hereditary target detection.Identify that specific genetic targets target MIP (MIP1-1) can be combined with the 2nd MIP (MIP1-2) of the same genetic targets target different piece of identification.This process can be repeated, produce and identify the many MIP of same genetic targets target (showing MIP1-50).Similarly, a collection of MIP can be produced to identify the second hereditary target (MIP2-50).These MIP may be used for analyzing, as shown in Figure 3, to compare two kinds of hereditary targets.
Connection described herein, hold lock or other oligonucleotide probe can mix with genomic dna.In some cases, use multiple oligonucleotide probe, comprise for the specific site on item chromosome or coloured differently body more than 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,100,200,500,1000,5000 or 10,000 kind of oligonucleotide probe.
Fig. 8 depicts the LDR-PCR using the oligonucleotide of multiplex to carry out in droplet, detects genetic targets target sensitivity in droplet to strengthen this method.As shown in Figure 7, a pair linear oligonucleotide (LDR1-1) is designed to identify genetic targets target adjacent area.Different identifies the second hereditary target to (LDR2-1).Multipair oligonucleotide (LDR1-50 and LDR2-50) can be designed to identify genetic targets target different piece.These oligonucleotide to a part of in conjunction with genetic targets target, and carry out connection as described in Figure 7.Use two kinds of hereditary targets of difference shown in two kinds of distinct colors detections.Such as, half LDR probe identifiable design target sequence is as suspicious dysploidy karyomit(e), and second half identifies the diploid karyomit(e) of canonical sequence as supposition, thus makes dysploidy detect the sensitivity with improvement.
In some embodiments, can increase in advance target and canonical sequence before the analysis using digital droplet to detect.The method of amplification is known in the art, comprises the sequencing reaction of maintenance automatically, ligase chain reaction, cDNA end rapid amplifying, polymerase chain reaction and ligase chain reaction, the amplification of Q phagus beta, strand displacement amplification, isothermal duplication or montage overlap extension pcr.Then the product increased in advance can use in method of the present invention.
Heredity target
In some embodiments, can to DNA or the RNA process of extracting to select, to mark, to catch and/or to be separated target sequence polynucleotide, this especially may comprise hereditary target described herein.In some cases, catch and to relate to from a large amount of genetic material physical sepn target sequence with being separated and removing unwanted genetic material.In some cases, by by the sequence of expectation be fixed on solid structure and realize physical sepn as the complementary sequence hybridization on the surface of polymer surfaces, polymer bead, magnetic micro-beads or microfluidic channel.In other cases, physical sepn is realized by affine method, as adopted the sequence of catching expectation with the probe of the complementary sequence of affinity labelling coupling, affine interactional non-limiting example comprises binding affinity and is greater than micromole, nmole, picomole, flies mole or be greater than the interaction of the Streptavidin-vitamin H, antibody-antigene, enzyme-substrate, receptor-ligand and the protein-small molecule that fly mole strength.After catching, in some cases, with washing methods well known in the art, the sequence of expectation can be separated from a large amount of genetic material, comprise with the buffer salt solution washing containing gentle ionic or non-ionic detergent, proteinase inhibitor and DNase inhibitor.In some embodiments, droplet described herein does not comprise microballon, polymer bead or magnetic bead.
Test described herein and the target of probe can be relevant any hereditary targets abnormal to fetus genetic, comprise dysploidy and other heritable variation, as sudden change, insertion, interpolation, disappearance, transposition, point mutation, Trinucleotide repeat diseases and/or single nucleotide polymorphism (SNP), and irrelevant contrast target with fetus genetic is abnormal.Also illustrate irrelevant other with fetus dysploidy herein to test.
Usually, method and composition described herein can allow to detect the karyomit(e) having more or lose, especially typically to inborn defect or miscarry those karyomit(e)s relevant.Such as, method and composition described herein allows to detect euchromosome trisomy (such as, 13 3 bodies, 15,16,18,21 or 22).In some cases, trisomy may to the abortion rate relevant (such as, 15 3 bodies, 16 or 22) increased.In other cases, detected trisomy to indicate the fetus of birth to have the life birth trisomy (such as, 13 3 bodies (Patau's syndrome), 18 3 bodies (Edward's syndrome) and trisomy 21 (mongolism)) of inborn defect.Extremely also may be the exception of sex chromosome (such as, XXY (klinefelter syndrome), XYY (jacob's syndrome) or XXX (trisomy X)).In some preferred embodiment, hereditary target is the one or more targets on one or more following karyomit(e): 13,18,21, X or Y.Such as, hereditary target can be 50 sites on karyomit(e) 21 and/or 50 sites on karyomit(e) 18 and/or 50 sites on karyomit(e) 13.
Other fetal disorder can determined based on method and system herein comprises one or more chromosomal monosomy (X chromosome monosomy, be also referred to as Turner syndrome), one or more karyomit(e) (13, 18, 21 and X) trisomy, (it is the most often observe in sex chromosome in the mankind for one or more chromosomal tetrasomy and five body constituents, such as, XXXX, XXYY, XXXY, XYYY, XXXXX, XXXXY, XXXYY, XYYYY and XXYYY), haploidy, triploidy (every bar karyomit(e) three, such as, 69 karyomit(e)s in the mankind), tetraploidy (every bar karyomit(e) four, such as, 92 karyomit(e)s in the mankind), pentaploidy and polyploidy.
In some cases, described hereditary target comprise on specific karyomit(e) more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 1, 000, 5, 000, 10, 000, 20, 000, 30, 000, 40, 000, 50, 000, 60, 000, 70, 000, 80, 000, 90, 000 or 100, 000 site.In some cases, described hereditary target is included in more than the target on 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 or 22 coloured differently body.In some cases, described hereditary target is included in the target be less than on 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22 or 23 coloured differently bodies.In some cases, hereditary target comprises the known gene suddenlyd change in the genetic diseases of heredity, comprises autosomal dominant illness and recessive disorder and sex-linked dominant illness and recessive disorder.Nonrestrictive example comprises the genetic mutation causing autoimmune disorder, neurodegenerative disease, cancer and metabolic disease.In some embodiments, the method by with reference in the hereditary target relevant to gene unconventionality and the hereditary target (as be positioned at normal diploid karyomit(e) on gene) irrelevant with gene unconventionality compare, and detect the existence of the hereditary target (as trisomy) relevant to gene unconventionality.
Method herein or composition also can comprise primer sets for chromosomal different zones and/or probe.Such as, multiple probe (such as, MIP probe, linking probe) can comprise at least one for the first probe of the first specific region in karyomit(e) and at least one the second probe for the second specific region in karyomit(e).In some cases, first probe signaling molecule or reagent (such as fluorophore) mark, the second probe second signal molecule (such as having the fluorophore with the diacritic color/wavelength of the color/wavelength of the fluorophore being coupled to the first probe) marks.Then described multiple probe can be incorporated into target polynucleotide.In selection scheme (such as, connection, cyclisation and Exonucleolytic ferment treatment etc. subsequently) after, the probe selected is assigned in multiple subregion (such as droplet), and subsequent analysis comprises the number of the subregion (such as droplet) of selected probe.Then target polynucleotide be can evaluate by the ratio between the number of the first probe and the number of the second probe and excalation, transposition or amplification whether comprised.Such as, can detect chromosomal excalation in this way, its middle probe 1 is for complete karyomit(e), and probe 2 is for the sequence in chromosomal lack part.In some embodiments, can use for different target more than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 kind of probe.In some cases, this number may more than 20,30,40,50,100,500,1000,5000,10000,50000,100000,500000,1000000 or more.
In a non-limiting example, the first linking probe (or primer sets) is for the q arm of karyomit(e) 21, and the second linking probe (or primer sets) is for p arm.If the result that both provide is each other reasonably close to (such as in some predetermined fiducial intervals), this can verify the measured value of karyomit(e) 21 concentration.On the other hand, if the measured value of target is obviously different with the measured value of those targets on q arm on p arm, this can show part dysploidy (chromosomal section), or can show that these test needs are further optimized or checking.
A kind of color for 21q target and the another kind of color for 21p target can be used to carry out the practical measurement of target group simultaneously.In addition, sample can be divided into two parts, in a part, carry out 21q mensuration, and carry out 21p mensuration in another part.Karyomit(e) also can be assigned to more than two primer sets (or oligonucleotide probe) to realize the meticulousr evaluation to chromosomal copy number.
Term " polynucleotide " refers to any nucleic acid molecule comprised more than a Nucleotide, can include but not limited to the length of 2,3,5,10,20,30,50,100,200,300,400,500 or 900 Nucleotide or 1,2,3,5,10,20,30,50,100,200,300,400,500 or 900 kilobase or 1,2,3,5,10,20,30,50,100,200,300,400,500 or 900 megabases.Polynucleotide also can refer to the coding region of gene, or the non-coding region of DNA, or whole karyomit(e).
" allelotrope " used herein occupies one of the gene of same position on karyomit(e) or several alternative forms of DNA non-coding region.Term " allelotrope " may be used for describing the DNA from any biology, includes but not limited to bacterium, virus, fungi, protozoon, mould, yeast, plant, the mankind, non-human, animal and archeobacteria.Such as, bacterium typically has a length dna chain.Term " allelotrope " about DNA of bacteria refers to compared with the form of homologous genes in different bacterium cell in same species, the form of this gene found in a kind of cell.
The alternative form (such as allelotrope) of gene can comprise the single nucleotide polymorphism (SNP) of one or more mononucleotides multi-form change.The alternative form of gene or non-coding region can comprise the adjacent repeat pattern of two or more Nucleotide, Short tandem repeatSTR (STR).
Allelotrope can have identical sequence or can have a Nucleotide or the change more than a Nucleotide.For the biologies with chromosomal two copies of every bar, if two karyomit(e)s all have identical allelotrope, this situation is called as isozygotys.If allelotrope on two karyomit(e)s is different, this situation is called as heterozygosis.
The example that target sequence exists the disease of a copy in fetus (homozygous) mother body D NA (heterozygous) disease comprises sickle cell's cystic fibrosis, hemophilia and the Qin-sachs' disease.Therefore, by method described herein, can distinguish and at specific site, there is the genome of a specific sudden change and there is at specific site the genome of two specific sudden changes.
Sicklemia is autosomal recessive disease.The Black American of 9% is heterozygosis, and 0.2% is homozygous recessive.Recessive alleles causes the amino-acid substitution in the β chain of oxyphorase.
The Qin-sachs' disease is the neural degenerative that autosomal recessive produces.Manifest symptom after birth.The children of this equipotential gene pure recessiveness seldom live through five years old.Patient lacks the ability producing N-acetyl-hexosaminidase, this enzyme liberating GM2 Sphingolipids,sialo.
Another example is pku (PKU), a kind of recessive hereditary disorder, and its patient lacks the ability that phenylalanine is converted into the enzyme of tyrosine by synthesis.The individuality of this equipotential gene pure recessiveness accumulates phenylalanine and abnormal degraded product in urine and blood.
Hemophilia is one group of disease that blood cannot normally condense.The factor in blood participates in blood coagulation.The haemophiliac lacking normal Factor VIII is called as and suffers from hemophilia A, and the patient lacking factors IX suffers from hemophilia B.These genes are carried on X chromosome, and therefore, primer and probe can in the method for detecting the defective type X chromosome or the normal allele of its father that fetus has been hereditary mother.
In some cases, hereditary target is the part of gene or gene, such as CFTR, Factor IX (F8 gene), beta globin, hemochromatosis, G6PD, multiple neurofibromatosis, GAPDH, amyloid beta or pyruvate kinase.
In some embodiments, hereditary target is the sequence that the variation of any copy number is relevant to disease or illness.The Other diseases caused by genetic abnormality comprises fetal rickets, the adrenoleukodystrophy that X-is chain, the Agammaglobulinemia that X-is chain, Alagille syndrome, the mental retardation syndrome that α-thalassemia X is chain, alzheimer's disease, alzheimer's disease, early onset familial amyotrophic lateral sclerosis, androgen-insensitivity syndrome, Angelman syndrome, ataxia syndrome (AtaxiaOverview), hereditary ataxia telangiectasia, Bake muscular dystrophy (being also dystrophic disease), Beckwith-Wiedemann syndrome (Beckwith-WiedemannSyndrome), β-thalassemia, biotinidase deficiency, BOR, BRCA1 and BRCA2 hereditary breast cancer/ovarian cancer, mammary cancer, CADASIL, Canavan is sick, cancer, Xia Ke-Mali-Tu Si inherited neurological pathology, 1 type Xia Ke-Mali-Tu Si DPN, 2 type Xia Ke-Mali-Tu Si DPN, 4 type Xia Ke-Mali-Tu Si DPN, X-type Xia Ke-Mali-Tu Si DPN, Cockayne syndrome, colorectal carcinoma, contractural arachnodactyly (ContracturalArachnodactyly), congenital craniosynostosis syndrome (FGFR is correlated with), cystic fibrosis, cystinosis, deafness and heredity hearing loss, DRPLA (dentatorubral-pallidoluysian atrophy), diGeorge's syndrome (being also 22q11 deletion syndrome), the dilated cardiomyopathy that X-is chain, mongolism (trisomy 21), duchenne muscular dystrophy (being also dystrophic disease), myodystonia, early onset myodystonia (DYT1), dystrophic disease, , Ehlers-Danlos syndrome, Kypho scoliosis type, vascular type Ehlers-Danlos syndrome, epidermolysis bullosa simplex, hereditary multiple exostoses, FSHD, factor Ⅴ Leiden thrombosis, familial adenomatous polyposis (FAP), familial Mediterranean fever, fragile X mental retardation, Friedreich ataxia, Frontotemporal dementia companion parkinsonism-17, galactosemia, gaucher's disease, hereditary hemochromatosis thesaurismosis, hemophilia A, hemophilia B, hemorrhagic telangiectasia heredity 55, non-syndrome hearing loss and deaf DFNA (connexin 26), non-syndrome hearing loss and deaf DFNB1 (connexin 26), hereditary spastic paraplegia, Hermansky-Pudlak syndrome, hexosaminidase A deficiency disease (the Ye Ji Qin-sachs' disease), Huntington Chorea, fetal rickets, recessive hereditary congenital ichthyosis, incontinentia pigmenti, Kennedy disease (being also that spinal cord and bulbar muscular atrophy are sick), galactosylceramide beta-galactosidase deficiency, leber hereditary optic neuropathy becomes, Lesch-Nychan syndrome leukemia, Li-Fo Meini syndrome, limb-waist muscular dystrophy, familial lipoprotein lipase deficiency, congenital agyria, Marfan's syndrome, MELAS (mitochondrial encephalomyopathy, lactic acidosis and the outbreak of palsy sample) monosomy, 2 type muitiple endocrine neoplasms, multiple exostoses, congenital hereditary muscular dystrophy, steirert-Batten-Gibb syndrome, nephrogenic diabetes insipidus, neurofibroma 1, neurofibromatosis 2, the inherited neurological pathology of easy trouble compression palsy, C type niemann-Pick disease, Nijmegen breakage syndrome, atrophia bulborum hereditaria, 1 type oculocutaneous albimism, oculopharyngeal muscular dystrophy disease, ovarian cancer, Pallister-Hall syndrome, handkerchief metal type teenager Parkinson's disease, PelizaeusMerzbacher is sick, Pendred syndrome, Peutz-Jeghers syndrome, phenylalanine hydroxylase deficiency is sick, Prader-Willi syndrome, the compound pituitary hormone deficiency (CPHD) that PROP-1 is relevant, prostate cancer, retinitis pigmentosa, retinoblastoma, rood Mu De-thomson syndrome, Smith-Lemli-Opitz syndrome, hereditary spastic paraplegia, spinal cord and bulbar muscular atrophy (being also Kennedy disease), spinal muscular atrophy, 1 type spinocebellar ataxia, 2 type spinocebellar ataxias, 3 type spinocebellar ataxias, 6 type spinocebellar ataxias, 7 type spinocebellar ataxias, Stickler syndrome (heredity arthro-ophthalmopathy), the Qin-sachs' disease (being also GM2 gangliosidosis) trisomy, plyability tuberous sclerosis, I type Usher syndrome, II type Usher syndrome, velo-cardio-facial syndrome (being also 22q11 deletion syndrome), VonHippel-Lindau syndrome, williams syndrome, Wilson is sick, the chain adrenoleukodystrophy of X, X-linked agammaglobulinemia, X-expander chain type myocardosis (being also dystrophic disease) and the chain hypotonic phase amentia syndrome of X-.
The generation of droplet
The disclosure comprises the composition and the method that detect fetal genetic material with droplet digital pcr.Droplet described herein comprises as U.S. Patent number 7,622, the droplet that the emulsion composition (or mixture of two or more immiscible fluid) described in 280 and the equipment described in international application no PCT/US2009/005317 as on September 23rd, 2009 submits to, the first invention people is Colston produce.Term " emulsion " used herein refers to the mixture of immiscible fluid (as oil and water).Oil phase and/or water-in-oil emulsion allow the compartmentation of water-based droplet reaction mixture.In preferred embodiments, emulsion comprises the water-based droplet in oil-continuous phase.In other cases, emulsion provided herein is oil-in-water emulsion, and wherein droplet is the oil droplet in continuous aqueous phase.Droplet provided herein is designed to prevent from mixing between compartment, and each compartment prevents its content from evaporating and prevents from merging with the content of other subregion.
Mixture described herein or emulsion may be stable or instability.In preferred embodiments, emulsion is relatively stable and seldom merge.When little droplet combines and forms larger droplet progressively, merge.In some cases, from droplet producer produce droplet be less than 0.00001%, 0.00005%, 0.00010%, 0.00050%, 0.001%, 0.005%, 0.01%, 0.05%, 1%, 5%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9% or 10% and other droplet merge.Emulsion also can have limited flocculation, and this is the process producing disperse phase with thin slice from suspension.
Be divided into by sample little reaction volume that the consumption of reagent can be made to reduce as described here, thus reduce the material cost analyzed.The dynamics range that sample complicacy also improves detection is reduced by distributing, because more abundant molecule and low-abundance molecule are in different subregions, thus make more low-abundance molecule with higher ratio contact reacts reagent, which in turn increases the detection of lower abundant species.
In some cases, the droplet that mean diameter is about 0.001,0.01,0.05,0.1,1,5,10,20,30,40,50,60,70,80,100,120,130,140,150,160,180,200,300,400 or 500 micron can be produced.The microfluidic methods that the cross-flow focusing of known microchannel or physical agitation produce emulsion droplets produces single dispersing or polydispersion emulsion.In some embodiments, droplet is single dispersing droplet.In some cases, produce droplet and make the size variation of described droplet be no more than the mean size of described droplet ± 5%.In some cases, produce droplet and make the size variation of described droplet be no more than the mean size of described droplet ± 2%.In some cases, droplet producer produces a group droplet from single increment product, wherein do not have the size variation of droplet exceed the mean size of the total group of droplet ± 0.1%, 0.5%, 1%, 1.5%, 2,2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.
Higher mechanical stability can be used for microfluidic procedures and more high-shear fluid handling (such as, turning as valve in microfluidic capillary or by 90 degree in fluid passage).The droplet of front thermal treatment and postheat treatment or capsule for standard pipetting and centrifugal be mechanically stable.
In some cases, flow through aqueous sample by oil phase and form droplet.In some preferred embodiments, aqueous phase comprises buffered soln for implementing PCR reaction and reagent, comprises Nucleotide, primer, probe, template nucleic acid, archaeal dna polymerase and optional reversed transcriptive enzyme for fluoroscopic examination.
In some cases, water-based droplet comprises for carrying out without solid-state microballon as the buffered soln of magnetic micro-beads and reagent.In some cases, buffered soln can comprise about 1,5,10,15,20,30,50,100 or 200mMTris.In some cases, the concentration of Repone K can be about 10,20,30,40,50,60,80,100,200mM.In preferred embodiments, buffered soln comprises 15mMTris and 50mMKCl.In some cases, Nucleotide comprises deoxyribonucleoside triphosphate molecule, comprises dATP, dCTP, dGTP, dTTP, and respective concentration is about 50,100,200,300,400,500,600 or 700 μMs.In some cases, dUTP is added in aqueous phase and be about 50,100,200,300,400,500,600,700,800,900 or 1000 μMs to concentration.In some cases, by magnesium chloride (MgCl
2) add in aqueous phase and be about 1.0,2.0,3.0,4.0 or 5.0mM to concentration.In a preferred embodiment, MgCl
2concentration be 3.2mM.
Non-specific blocking agent can be used as BSA or the gelatin from ox-hide skin, and wherein the concentration range of gelatin or BSA is about 0.1-0.9%w/v.Other possible encapsulant can comprise beta lactoglobulin, casein, milk powder or other conventional encapsulant.In some cases, the preferred concentration of BSA and gelatin is 0.1%w/v.
Primer for increasing in aqueous phase can have the concentration being about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0 or 1000 μM.In a preferred embodiment, the concentration of primer is 0.5 μM.In some cases, aqueous phase comprises one or more probes for fluoroscopic examination, and its concentration is about 0.1,0.2,0.3,0.4 or 5.0 μM.In a preferred embodiment, the concentration for the probe of fluoroscopic examination is 0.25 μM.In PCR, the proper range of target nucleic acid concentration is about 1pg to about 500ng.
In some embodiments, aqueous phase also can comprise additive, include but not limited to non-specific background/locked nucleic acids (such as, salmon sperm DNA), biological preservative (such as sodium azide), PCR toughener (such as, trimethyl-glycine, trehalose etc.) and inhibitor (such as RNAse inhibitor).
In some cases, add in aqueous phase by the ethylene oxide/propylene oxide segmented copolymer of non-ionic type, its concentration is about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%.Conventional bio-surfactant comprises nonionic surface active agent as PluronicF-68, Tetronics, ZonylFSN.In a preferred embodiment, the concentration of PluronicF-68 is 0.5%w/v.
In some cases, the concentration that magnesium sulfate can be similar replaces magnesium chloride.Multiple conventional commercially PCR damping fluid from different supplier can replace buffered soln.
Oil phase can comprise the base oil fluoridized, and the latter is stablized extraly by combining as perfluorinated polyether with fluorinated surfactant.In some cases, base oil can be one or more in HFE7500, FC-40, FC-43, FC-70 or other conventional fluorinated oil.In some cases, aniorfic surfactant is the morpholino derivative of Krytox ammonium (Krytox-AM), KrytoxFSH ammonium salt or KrytoxFSH.The concentration of Krytox-AS can be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% or 4.0%w/w.In some preferred embodiments, the concentration of Krytox-AS is 1.8%.In other preferred embodiment, the concentration of Krytox-AS is 1.62%.The concentration of the morpholino derivative of KrytoxFSH can be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% or 4.0%w/w.In some preferred embodiments, the concentration of the morpholino derivative of KrytoxFSH is 1.8%.In some preferred embodiments, the concentration of the morpholino derivative of KrytoxFSH is 1.62%.
Oil phase can comprise further for regulating oily characteristic as vapour pressure or viscosity or capillary additive.Nonrestrictive example comprises perfluor-octanol and 1H, 1H, 2H, 2H-perfluor decyl alcohol.In some preferred embodiments, add 1H, 1H, 2H, 2H-perfluor decyl alcohol to concentration is about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, 2.75% or 3.00%w/w.In some preferred embodiments, add 1H, 1H, 2H, 2H-perfluor decyl alcohol to concentration is 0.18%w/v.
In some embodiments, preparation emulsion is to produce the height single dispersing droplet with liquid-like interfacial film, and it can be the microcapsule with solid-like interfacial film by thermal conversion; Such microcapsule can be used as bio-reactor by reaction process as pcr amplification can keep its content.The conversion to microencapsulation form can be there is after heating.Such as, this conversion can occur at higher than the temperature of about 50,60,70,80,90 or 95 degrees Celsius.In some cases, this heating thermal cycler carries out.During heating, fluid or mineral oil can be used to cover to avoid evaporating.Before heating, can remove or excessive oil-continuous phase can not be removed.Biocompatible capsule can be resisted and merge and/or flocculation in numerous heating and mechanical treatment.
After conversion, capsule can be stored in about 3,4,5,6,7,8,9,10,15,20,25,30,35 or 40 degree, and a preferred embodiment is included in and stores capsule lower than about 25 degree.In some embodiments, these capsules can be used for biomedical applications, as macromolecular stable digitizing encapsulation, especially comprise the aqueous biological fluids of nucleic acid or protein or the mixture both this; Medicine and vaccine delivery; Library of biomolecules; Clinical imaging is applied and other.
Microcapsule can comprise one or more of nucleic acid probe (such as, molecular inversion probes, linking probe etc.), and can resist merging, especially at high temperature.Therefore, pcr amplification reaction can carry out under very high density (such as, the stoichiometric number of each unit volume).In some cases, every ml can occur more than 100, and 000,500,000,1,000,000,1,500,000,2,000,000,2,500,000,5,000,000 or 10,000,000 independent reaction.In some cases, in single hole, such as, react in a hole of microtiter plate, without intersecting mixing between reaction volume.Microcapsule also can comprise makes PCR react generation other composition necessary, such as primer, probe, dNTP, DNA or RNA polymerase etc.These capsules show the resistance being combined and flocculating in numerous heat and mechanical treatment.
The effect of device
Multiple device can be used to realize method described herein.Fig. 3 shows the schema of the illustrative methods for diagnosing fetal dysploidy, and highlights some devices that can use in method herein.Obtain the maternal tissue's sample (201) comprising mother and fetal genetic material.From sample, extract DNA, be incorporated into the probe identifying karyomit(e) 1 (202) and 21 (203), then carry out ligation.The sample of the probe (and the component needed for PCR reaction) comprising connection is added droplet maker (301), and probe is dispensed in the multiple droplets in water-in-oil emulsion by the latter.The example of some useful in this disclosure droplet makers on September 23rd, 2009 submit to, provide in international application no PCT/US2009/005317 that the first invention people is Colston.Then littlely the amplification hatching to allow probe in thermal cycler (302) is dropped in.During amplified reaction, relative to not containing the droplet of the probe of amplification, comprise the fluorescence increase of the droplet of the probe of amplification.Then make droplet individually by droplet reading apparatus (303) process, collect data to detect fluorescence.The example of some useful in this disclosure droplet readers on September 23rd, 2009 submit to, provide in international application no PCT/US2009/005317 that the first invention people is Colston.
As shown in Figure 3, then the data of the copy number about karyomit(e) 1 and 21 are compared to detect fetus dysploidy.Usual use by device as these data of the Algorithm Analysis of computer utility.In some cases, droplet maker, thermal cycler, droplet reader and computer are the devices separated separately.In other cases, device comprises such device of two or more arbitrary combination.Such as, the device thermal cycler that can comprise droplet maker and be communicated with it.In other cases, a device may comprise droplet maker, thermal cycler and droplet reader.
The disclosure provides the method for detecting copy number and/or detection copy number variation (such as, fetus dysploidy) fast, effectively and delicately.The disclosure especially can be used for identifying in genetic material with the copy number change of the polynucleotide (the fetus polynucleotide in such as maternal blood sample) of minute quantity existence.In some cases, the target polynucleotide being less than 0.00001,0.00005,0.00010,0.00050,0.001,0.005,0.01,0.05,0.1,0.5,1,2,2.5,3,3.5,4,4.5,5,6,7,8,9 or 10 copy is detected.In some cases, the target polynucleotide being less than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,150,200,250,300,350,400,450 or 500 copy is detected.In some cases, droplet described herein is produced with the speed being greater than 1,2,3,4,5,10,50,100,200,300,400,500,600,700,800,900 or 1000 droplets per second.
Amplification
Technology for increase target and canonical sequence (and the sequence in linking probe) is known in the art, comprises U.S. Patent number 7,048, the method described in 481.In brief, this technology comprises method and composition sample being divided into little droplet, in some cases, each droplet separately average packet containing being less than a nucleic acid molecule, the nucleotide sequence increased in each droplet, and the existence detecting particular target sequence.In some cases, the sequence be amplified is present on the probe for genomic dna, instead of genomic dna originally with it.
According to known parameter designing primer to avoid secondary structure and self hybridization.In some embodiments, different primer pairs with roughly the same annealing temperature and unwinding, such as the annealing of another primer pair and melting temperature(Tm) ± scope of 1,2,3,4,5,6,7,8,9 or 10 DEG C in.In some cases, only attachable probe is added in genomic dna at first, and do not add primer, distribute the probe of connection subsequently, with such as universal primer, the one or more bar sequences on each subregion internal probe are increased subsequently.In some cases, at first to use more than 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,100,200,500,1000,5000,10,000 or more bar probes.Such probe possibility can with hereditary target hybridization described herein.Such as, can use the mixture of probe, wherein at least one probe is for the specific site on karyomit(e), and the second probe is for the different loci on same karyomit(e) or different karyomit(e).Often group can have himself general probe group by linking probe, and is distinguished by the corresponding TaqMan probe often organized.Or all linking probe group can use identical universal primer group, and distinguished by the corresponding TaqMan probe often organized.The exemplary sequence with human gene group DNA without the general probe of homology comprises SEQIDNO:79 and 80 (Figure 17).
Although describe the preferred embodiments of the invention according to PCR, the present invention relates generally to the application that multiple single genetic sequence detects.In some embodiments, amplification method can be, such as, the sequencing reaction automatically maintained, ligase chain reaction, cDNA end rapid amplifying, polymerase chain reaction, the amplification of Q phagus beta, strand displacement amplification, isothermal duplication or montage overlap extension pcr.
Primer can be prepared by multiple method, include but not limited to clone suitable sequence and direct chemosynthesis sequence (Narang etc., MethodsEnzymol.68:90 (1979) by method well known in the art; Brown etc., MethodsEnzymol.68:109 (1979)).Also primer can be obtained from commercial source, as OperonTechnologies, AmershamPharmaciaBiotech, Sigma and LifeTechnologies.Primer can have identical melting temperature(Tm).The length of primer can extend or shorten to produce the primer with the melting temperature(Tm) of expectation at 5 ' end or 3 ' end.In preferred embodiments, a primer in primer pair is longer than another primer.In preferred embodiments, 3 ' annealing length of the primer in primer pair is different.Also the annealing position of each primer pair can be designed so that the sequence of primer pair and length produce the melting temperature(Tm) expected.For determining that the most simple equation of the melting temperature(Tm) of the primer being less than 25 base pairs is Wallace's rule (WallaceRule) (Td=2 (A+T)+4 (G+C)).Also computer programming primer be can use, ArrayDesignerSoftware (ArrayitInc.), the sequence oligonucleotide probe design software (OlympusOpticalCo.) for genetic analysis, NetPrimer and the DNAsis from HitachiSoftwareEngineering included but not limited to.With software program as NetPrimer (based on the program of free webpage, http://premierbiosoft.com/netprimer/netprlaunch/netprlaunch.htm l; The internet address on August 17th, 2002) calculate the TM (unwinding or annealing temperature) of every bar primer.In another embodiment, after the amplification of any circulation, include but not limited to circulation 1,2,3,4,5, circulation 6-10, circulation 10-15, circulation 15-20, circulation 20-25, after circulation 25-30, circulation 30-35 or circulation 35-40, can recalculate and improve the annealing temperature of primer.After initial amplification cycles, 5 ' half of primer is merged in the product of each interested locus, therefore can recalculate TM based on the sequence of 5 ' one half-sum 3 ' half of every bar primer.
In some preferred embodiments, template MIP represents the sequence of the expectation comprising target and canonical sequence, and template MIP is separated as mentioned above and the MIP of linearizing previous cyclisation.Template MIP in PCR as template molecule.In some cases, before droplet generates, produce template MIP, in other cases, between droplet generation or afterwards, produce template MIP.In the example of latter event, in the unzipping step of the PCR reaction in thermal cycler after heating, uridylic-N-de-glycosylation ferment treatment urine pyrimidine alkali base causes cyclisation MIP to contain abasic site, and such cyclisation MIP experiences spontaneous ring-opening reaction.In some cases, template MIP is as the DNA profiling of droplet digital pcr, and wherein the amplification of template MIP corresponds to the detection of the expectation sequence (such as, mark or canonical sequence) representated by MIP.In some embodiments, the method relates to the droplet producing by being flowed in sample fluid by immiscible liquid and be used for the reaction of droplet digital pcr, and wherein sample fluid comprises one or more MIP or one or more template MIP and comprises the main mixture of the required reagent of PCR.In some preferred embodiments, the main mixture of PCR comprises thermostable polymerases, for the universal primer of template MIP amplification, for the dissociative DNA Nucleotide that combines and the buffer composition for reacting.When being exposed to higher than 99,98,97,96,95,94,93,92,91,90,80,70 degree or lower temperature, thermostable polymerases can keep active.In some cases, sample fluid also comprises the genomic dna of digestion or the enzyme of deactivation as endonuclease and/or the deglycosylating enzyme that retains in MIP template produces.In some embodiments, present method relate to produce comprise be less than 1,1 or more than 1 genome equivalent by the droplet of the DNA of MIP or MIP template representative.
In some embodiments, the sequence that can comprise the expectation of target and canonical sequence is present in the mixture containing less desirable background genes group DNA as a part.In some cases, joint detection reaction and droplet digital pcr only detect the sequence of expectation and cannot detect that background genes group DNA sequence dna (such as, comprise in the PCR of main mixture of universal primer in use, only connect product and can be formed and can detect product).In other cases, in droplet digital pcr, the sequence of expectation is detected with sequence specific primers.
In some preferred embodiments, the disclosure relates to the composition comprising emulsion, comprises the DNA that can be used for detecting fetal genetic material of average about 1 genome equivalent in this emulsion.In some cases, the interested sequence of one or more MIP or MIP template representative (region as karyomit(e) 21), its detection can help to determine fetus dysploidy.In some cases, the composition of the genetic targets target sequence interested that representative may be relevant with genetic abnormality (as trisomy) will can be comprised compared with comprising the composition of the sequence representing the canonical sequence that may have nothing to do with genetic abnormality.In some cases, by can strengthen the sensitivity of detection for the multiplex of genetic targets target probe.And detecting pattern while of using multiple, the different colours in fluorescence detection method as detailed below, can detect multiple hereditary target abreast.
In some embodiments, hereditary target can comprise any nucleic acid molecule that can be connected product representative, as the probe of MIP, MIP template or connection.These connect product and are present in sample fluid, and immiscible liquid flows into wherein to produce droplet.Also the required reagent of PCR can be comprised, for droplet digital pcr subsequently in droplet.Analyzable genetic targets target example comprises heritable variation that may be irrelevant with fetus gene unconventionality herein, as dysploidy, sudden change, insertion, interpolation, disappearance, transposition, point mutation, Trinucleotide repeat diseases and/or single nucleotide polymorphism (SNP).
After any amplification cycles, include but not limited to circulation 1,2,3,4,5, circulation 6-10, circulation 10-15, circulation 15-20, circulation 20-25, after circulation 25-30, circulation 30-35 or circulation 35-40, can recalculate and improve the annealing temperature of primer.After initial amplification cycles, 5 ' half of primer is merged in the product of each interested locus, therefore can recalculate TM based on the sequence of 5 ' one half-sum 3 ' half of every bar primer.Any archaeal dna polymerase of catalysis primer extension can be used, include but not limited to the Klenow fragment of e. coli dna polymerase, e. coli dna polymerase 1, T7DNA polysaccharase, T4DNA polysaccharase, Taq polysaccharase, PfuDNA polysaccharase, VentDNA polysaccharase, phage 29, REDTaq
tM, genomic dna polysaccharase or Sequenase.Preferably, heat-staple archaeal dna polymerase is used.Also can carry out heat start PCR, wherein reactant is heated to 95 DEG C before adding polysaccharase and keep 2 minutes, or polysaccharase can keep non-activity until first heating steps of the 1st circulation.Heat start PCR can be used to minimize to make non-specific amplification.Any PCR cycle number can be used to carry out DNA amplification, include but not limited to 2,5,10,15,20,25,30,35,40 or 45 circulations.
Can by any method amplification target nucleic acids (such as, linking probe, MIP probe) known in the art.In some cases, to be increased target nucleic acids by polymerase chain reaction (PCR).The example of operable round pcr includes but not limited to quantitative PCR, quantitative fluorescence PCR (QF-PCR), multiple fluorescence PCR (MF-PCR), PCR in real time (RT-PCR), singe-cell PCR, restriction fragment length polymorphism PCR (PCR-RFLP), PCR-RFLP/RT-PCR-RFLP, heat start PCR, nest-type PRC, original position polononyPCR, original position rolling circle amplification (RCA), bridge joint PCR, picotrace titration (picotiter) PCR and emulsion-based PCR.Other suitable amplification method comprises ligase chain reaction (LCR), transcription amplification, automatically maintains sequence replicating, the polymerase chain reaction (CP-PCR) that the selective amplification of target polynucleotide sequence, consensus sequence cause, the polymerase chain reaction (AP-PCR) caused arbitrarily, PCR (DOP-PCR) that degenerate oligonucleotide causes and the sequence amplification (NABSA) based on nucleic acid.Herein can other amplification method comprise United States Patent (USP) 5,242,794; 5,494,810; 4,988,617; With 6,582, those described in 938.In some embodiments, the amplification of target nucleic acids can occur on microballon.In other embodiments, amplification does not occur on microballon.
In some cases, thermal cycle reaction is carried out to the sample comprised in droplet.In some cases, during thermal cycling, droplet keeps complete.During thermal cycling, droplet can keep complete, and its density is higher than about 10,000/ml, 100,000/ml, 200,000/ml, 300,000/ml, 400,000/ml, 500,000/ml, 600,000/ml, 700,000/ml, 800,000/ml, 900,000/ml or 1,000,000/ml.In other cases, may merge at the droplet of thermal cycling period two or more.In other cases, more than 100 or more than 1 during thermal cycling, 000 droplet may merge.
Determination and analysis
Using fluorescence technology completes the detection of PCR primer.The DNA intercalative dye of Fluorescence Increasing is as Australia's second ingot or SYBR is green can provide quantitative reading to the DNA amount be present in reaction volume after in conjunction with DNA.Increase with reaction process because this DNA measures, fluorescence intensity also increases.The method relating to DNA intercalative dye is responsive to background fluorescence, because they are not measure DNA in sequence-specific mode, can not distinguish reaction product with other molecule as primer dimer.There is provided the method for sequence-specific detection PCR primer to relate to comprise fluorescent agent-quencher to and and the specific sequence probe of hybridizing.Fluorescent agent can be anyly send the molecule that can detect light, and as fluorophore, quencher can be this transmitting of any absorption thus reduce the molecule of fluorescent agent emissive porwer.When being present in the solution comprising complementary sequence, fluorescent agent-quencher probe is combined with sequence.Between the PCR reaction period, polysaccharase such as Taq can with this probe as primer, and 5 ' → 3 ' exonuclease activity working to excise RNA primer in cell cuts probe.Using the fluorescent agent-quencher probe of synthesis as in the PCR reaction of primer, 5 ' → 3 ' exonuclease activity makes probe be cut, and causes fluorescent agent to be separated with quencher.Once fluorescent agent is no longer covalently bound with quencher, its fluorescent emission can be detected.
The preferred embodiment of present disclosure relates to the droplet digital pcr product detecting and utilize MIP template to produce.In some cases, the probe combined by cutting and MIP specific sequence (being different from hereditary target) is detected.This strategy allows to use general fluorescent agent-quencher probe, this probe in detecting MIP, and does not need the genetic targets target sequence-specific for MIP representative.
In some embodiments, can use molecular beacon (MB) probe, it becomes and fluoresces after being incorporated into target sequence.MB probe is the oligonucleotide of stem-ring structure, and it comprises fluorescent agent at 5 ' end, comprises quencher at 3 ' end.6 powers of the quencher degree shifted by Fluorescence Resonance Energy and the distance between quencher and fluorescent agent are inversely proportional to.After heating and cooling, stem-ring structure transformed by MB probe, and this quencher is from the fluorescent signal of fluorescent agent.If there is the PCR primer of its sequence and ring complementary in heating/cooling cycle, the distance that the hybridization of a chain of MB and PCR primer will increase between quencher and fluorescent agent, causes Fluorescence Increasing.
In some embodiments, general probe is used to detect.General fluorescent probe (UFP) comprises fluorescence molecule, and this fluorescence molecule launches detectable electromagnetic radiation after the electromagnetic radiation of absorption one wavelength range.General quenching probe (UQP) comprises quenching molecules, and this quenching molecules reduces the fluorescent emission intensity of the fluorescent probe near it.In one case, universal fluorescent probe comprises nucleic acid segment, and the complementary nucleic acid section on this nucleic acid segment and general quenching probe or the complementary nucleic acid section (as MIP) in target sequence are hybridized.During PCR, the amplification of this target sequence causes universal fluorescent probe to strengthen with the combination of quenching probe compared with the combination of target sequence, and this causes detectable Fluorescence Increasing.In some cases, the length that can change complementary sequence between universal fluorescent probe and general quenching probe is with the melting temperature(Tm) of the mixture regulating universal fluorescent probe and be combined with general quenching probe.In some cases, the length of complementary sequence can be 15 base pairs.In some cases, the length of complementary sequence can more than about 5,10,15,20,25,30,35,40,45,50,55,60,70 or 80 base pairs.The melting temperature(Tm) of the mixture that universal fluorescent probe is combined with general quenching probe can higher than about 40,45,50,55,60,65,70,75,80,85,90 or 95 degrees Celsius.
Fig. 6 display, without the need to cutting, detects the two color system of the nucleic acid in droplet with universal primer and general probe.General probe comprises two complementary oligonucleotide, and a fluorescent probe comprises fluorescence molecule (UFP1 or UFP2), and a quenching probe comprises quenching molecules (UQP1 or UQP2).UFP1 and UFP2 sends the fluorescence of different colours, can distinguish in the detection.When being incorporated into quenching probe, the fluorescence intensity of fluorescent probe significantly reduces.In addition, two comprise the region with fluorescent probe complementation to general forward and reverse primer, and promote the pcr amplification of target sequence.In first round amplification, be merged in template by universal primer with the region of fluorescent probe complementation.Take turns in amplification subsequently several, therefore fluorescent probe UFP1 or UFP2 can hybridize with this template, instead of hybridize with their respective quenching probes.Due to by amplified reaction exponential create these templates more, UFP1-UQP1 and UFP2-UQP2 mixture is replaced by competitive binding by UFP1-template and UFP2 template composite.Separation fluorescent probe and quenching probe cause fluorescence intensity in reaction to strengthen, and can detect in the following steps.
By methods known in the art design general probe.In some embodiments, probe is stochastic sequence.General probe can be selected to ensure that it can not in test in conjunction with target polynucleotide, or other non-target polynucleotide that may exist in sample (such as, the extra-regional genomic dna of target polynucleotide).The exemplary sequence of general probe comprises SEQIDNO:81 and 82.
Fluoroscopic examination can be realized with being equipped with the multiple proofing unit of the module producing the exciting light that can be absorbed by fluorescent agent and the module detecting the light that fluorescent agent is launched.In some cases, sample (as droplet) can be detected in a large number.Such as, can by sample dispense to the plastics tubing being placed in detector, this detectors measure is from a large amount of fluorescence of plastics tubing.In some cases, a or more sample (as droplet) can be assigned to plate as in one or more holes of 96 holes or 384 orifice plates, can detect the fluorescence in each hole with fluorescence plate reader.
In some cases, detector comprises the ability of process droplet sample further, and wherein each droplet enters detector, detects, and then leaves detector.Such as, flow cytometry device can be improved to detect the fluorescence from droplet sample.In some cases, the fluorescence of microfluidic device detection from single file droplet of the pump be equipped with for controlling droplet movement can be used.In some cases, little dropping in two-dimensional surface is formed array, and detector moves relative to surface, is comprising each position detection fluorescence of single droplet.
In some cases, after obtaining fluoroscopic examination data, by computer stored and processing data.The executable logic of computer can be adopted to carry out these functions, as subtracting background fluorescence, appointment target and/or canonical sequence and quantitative data.Such as, count enable comprises corresponding to the suspection existed in sample the droplet number of the fluorescence being the karyomit(e) (as karyomit(e) 11) of dysploidy, and compared with comprising the droplet number of the fluorescence corresponding to the karyomit(e) (as karyomit(e) 1) not suspecting dysploidy existed.Fig. 9 shows a computer, and it is for showing, storing, retrieve or calculate the diagnostic result analyzed from molecular spectra; Display, storage, retrieval or the raw data calculated from genome or expression of nucleic acid analysis; Or display, storage, retrieval or calculating any sample useful in the method for the invention or patient information.
After digital pcr primer amplification target and canonical sequence are carried out to sample, the quantity of the quantity that can compare the positive with target sequence and the positive with canonical sequence.Because this compares the sequence existed in maternal tissue, do not need the DNA distinguishing mother and fetus.When target sequence be known as diplontic canonical sequence there is identical copies number time, so can determine that this sample is also diploid.When target sequence is different from canonical sequence, so this sample may comprise dysploidy.
In some embodiments, the genomic dna obtained in maternal tissue is from above assigned in multiple reaction volume (such as droplet), and each droplet is on average had be less than a genome equivalent (GE).In some cases, the average gene group equivalent that droplet comprises far more than a GE/ droplet, as 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,30,45 or 50GE/ droplet.In some cases, sample produces average more than 1,5 or 10GE/ droplet, but however, some droplets do not comprise GE or do not comprise target polynucleotide.In this case, may be necessary that application algorithm is to calculate specific genetic targets target copy mean number/droplet.In some cases, described hereditary target is actually complete karyomit(e) (or fragment), and it is then by fragmentation, and therefore a copy can appear in multiple droplet.
Usually, when analyzing single discrete reaction volume and whether there is genetic abnormality to be detected, DNA (chromosomal) to be analyzed is possibility or presence or absence on an average, thus allows so-called numerical analysis.The sum comprising the reaction volume of particular target sequence can compare with canonical sequence with the difference in display number.The ratio of improper between target sequence and the canonical sequence being known as diploid sequence (such as, 1: 1) shows dysploidy.Such as, sample can be assigned to reaction volume as in droplet, and makes often to drip droplet and comprise the DNA being less than a nominal genome equivalent.Can by checking a large amount of reaction volumes (such as, droplet), as 10,000,20,000,50,000,100,000,200,000,500,000 or more, determine the relative proportion of interested target (such as, for genetic marker or the unrelated probe of karyomit(e) trisomy 21) and canonical sequence (the known diploid sequence on such as karyomit(e) 1 or unrelated probe).In some cases, Target nucleotides (or genome equivalent)/droplet that reaction volume is as on average one or more in droplet comprises.In some cases, by application algorithm, as Dube etc. (2008) PlosOne3 (8): the algorithm described in e2876, calculates the average copy number of Target nucleotides.
By analyzing a large amount of reaction volumes, can measure from the mixture (wherein the relative concentration of foetal DNA is lower than mother body D NA) of the fetus initial sample and mother body D NA the change of relative proportion apart from 1: 1 caused by fetus dysploidy.This is called as numerical analysis, because each reaction volume on average has a genome equivalent/reaction volume, and extent of dilution can read as the binary result " be-no " that whether there is sequence (such as, target or reference) to be counted.
Method and composition described herein may be used in far-ranging application.In some embodiments, described method and composition relates to for diagnosing, detecting, identify, predict, evaluate or the method for situation that prognosis is relevant to inherited disorder.This situation may be due to genetic cause, comprises heredopathia, variation, sudden change, SNP, disappearance, amplification, transposition, inversion or other exception in specific gene seat (comprising any locus provided herein) any.
Method and composition provided herein can be used for diagnosing, detect, predict, identify or evaluate the risk that fetus has genetic abnormality (such as, mongolism, fetus dysploidy etc.).The method also can be used for identifying, quantitatively, diagnosis, prediction, assessment or analyze the risk that pregnant woman goes wrong when pregnancy, be included in the miscarriage in First Trimester, second trimester of pregnancy or third trimester of pregnancy; Stillborn foetus; The inborn defect of baby; Premature labor or other childbirth problem; And to conceived, give a birth or other situation any that baby due is relevant.
Method and composition provided herein can be used for the Relative copy number of evaluation first polynucleotide (such as DNA, RNA, genomic dna, mRNA, siRNA, miRNA, cRNA, single stranded DNA, double-stranded DNA, single stranded RNA, double-stranded RNA, tRNA, rRNA, cDNA etc.) relative to the second polynucleotide.Described method can be used for the amount of synthetic plasmid in analytical solution; Detect the sequence from the causal organism (such as bacterium, virus, retrovirus, slow virus, HIV-1, HIV-2, influenza virus etc.) the sample that individuality obtains.Described method also can be used for other to be existed in the application of few polynucleotide colony in compared with most nucleotide colony.
Some examples of method
In some embodiments, present method generally includes following steps:
1. the tissue comprising DNA is obtained from the individuality of pregnancy.In some embodiments, tissue can be the blood (whole blood or peripheral blood) of mother, blood plasma or serum.This material can be the blood gathered, and Circulating DNA is found in blood plasma, instead of in cell.In some embodiments, can by the foetal DNA in known method enrichment maternal tissue (as blood or blood plasma), as size fractionation is separated the DNA fragmentation to select to be less than about 300 Nucleotide.In some embodiments, the mother body D NA being usually greater than about 500 Nucleotide can be got rid of.In other embodiments, another enriching step formaldehyde treated blood sample can be utilized, as Dhallan etc. described in " MethodstoIncreasethePercentageofFreeFetalDNARecoveredFro mtheMaternalCirculation, " (2004) J.Am.Med.Soc.291:1114-1119.In other embodiments, by method well known in the art as alcohol settling purify DNA from other material sample.
Optionally, by specific binding one or more bar oligonucleotide or interested sequence in genomic dna can be caught by probe.In some cases, the mixture of genomic dna and probe is carried out ligase enzyme reaction, it causes the probe selectivity being incorporated into genomic dna to connect.In some cases, be combined cause linear probe as the inversion of MIP and cyclisation with DNA, described ligation produces cyclic products.In some cases, DNA and unconjugated probe can be removed with Exonucleolytic ferment treatment from sample.
2. a kind of DNA molecular from this sample is assigned to and is distributed by initial sample and in the discrete multiple reaction volumes (as aqueous phase droplet) obtained.In some embodiments, can the number of selective reaction volume to obtain the significance,statistical result of target copy number in initial sample DNA molecular.Reaction volume can be limited in small volume, and to make reaction molecular close proximity, this can Reaction time shorten.In each reaction volume, the order of magnitude of the amount of DNA molecular can be each reaction volume 0.5,1,2,3,4 or more DNA molecular.In some cases, in each reaction volume, the number of DNA molecular is on average about in each reaction volume and is about 1 DNA molecular.In some cases, the reaction volume of a droplet can as many as 100pL, 500pL, 1nL, 10nL or 100nL.
3. use nucleic acid amplification technologies as the existence of target sequence in PCR reaction detection DNA.Each reaction volume (such as droplet) can comprise all for carrying out the required reagent of PCR well known in the art.In one embodiment, by maternal tissue's sample dispense in droplet, increase this droplet in Continuous Flow pcr amplification reaction, as U.S. Patent number 7,048,481 and U.S. Patents Serial numbers 61/194, described in 043, both is intactly quoted all herein and is incorporated to.In some cases, amplification is the amplification to the sequence be incorporated in the probe of genomic dna, instead of the amplification of genomic dna itself.In other embodiments, by maternal tissue's sample dispense in droplet, increase this droplet in the hole of thermal cycler.In some embodiments, PCR primer can be detected or mark quantitatively to read easily.Such as, the fluorescent signal of one or more sequence in each reaction volume can be read, as the fluorescent signal by using fluorescent mark, probe or intercalative dye to produce.This detecting step is called as digital pcr herein, can be undertaken by multiple method, the fluorescence as by measuring following material: each droplet comprising PCR primer in the stream of the stream that (a) flows or stopping; B () is from the PCR primer of sample being diluted to each hole in microtiter plate; C () is from the PCR primer of the sample be diluted in emulsion; Or (d) carrys out the PCR primer of the sample of catching in comfortable microfluidic chamber; And
4. the quantitative analysis of the target sequence detection of couple mother and fetus.In some cases, this can comprise the target for different zones, as the probe for the target on karyomit(e), compared with the sequence on the normal diploid karyomit(e) being used as reference, suspect that the target on this karyomit(e) exists with abnormal copy number (as trisomy).This analysis also can relate to detecting and connects product, as being separated (such as, utilizing Exonucleolytic ferment treatment) with genomic dna and unconjugated probe and with the linearizing ring-type MIP of ferment treatment.In some cases, carry out quantitative assay by the fluorescence intensity measuring each subregion, and in other cases, measured by the number of partitions of counting containing detectable signal.In some embodiments, control sample can be comprised to provide the background measurements of the representative background fluorescence that can deduct from all observed values.In other embodiments, with 1,2,3,4 or can measure different sequence more than 4 kinds of different colours, such as, in different PCR primer, use the fluorophore of different colours, described PCR primer matches with the not homotactic probe of identification.
Embodiment
The double-colored detection scheme of embodiment 1. detects foetal DNA
In the present embodiment, describe the detection of trisomy 21 fetus dysploidy, in Maternal plasma sample, wherein contain the foetal DNA of 3%.Containing 1000 genome equivalents (GE)/mL in Maternal plasma, and gather the maternal blood volume of 20mL.
By centrifugal from maternal blood sample separated plasma, purification of nucleic acid is also concentrated into the volume of 50 μ L.Sample comprises the PCR reagent that multiplex measures component mix with isopyknic.By whole 100 μ L sample dispense to 100, in 000 dripping property droplet, often drip droplet and there is 1nL volume.The little titer per-cent of the desirable positive for 75%, this means the target sequence having 1.47 copies based on each droplet of Poisson's distribution, and this may be interpreted as needs by compartmentation to 100,147,000 target in 000 1nL droplet.The quantity of for this reason required primer sets is 147,000GE/10,000GE, i.e. 15 weights.Therefore, often dripping in droplet, having 15 weights for every bar target and canonical sequence, or often dripping droplet and always have 30 primer sets.With double-colored detection scheme analytic sample, wherein target sequence probe green emitter fluoresces, and canonical sequence probe is yellow, orange or red projector fluoresces.To more than 100,000 droplet detects, and calculates target (green) and the ratio with reference to (yellow, orange or red) sequence.
The monochromatic detection scheme of embodiment 2. detects foetal DNA
Use the condition of embodiment 1 herein, difference is it is not the targeting probe and the reference probe that use different colours, but separates sample (being such as divided into two), then produces two groups of droplets, amplification, then half targeting probe, second half with reference probe analyze respectively.
Embodiment 3. detects foetal DNA with MIP-ddPCR
From maternal blood sample, acellular blood plasma is separated by centrifugal.Then Cell-free DNA test kit (Qiagen) purifying is used and condensed nucleic acid.Then by the genomic dna of purifying and 1000 specific oligonucleotide probes of the chromosome sequence for karyomit(e) 21 (such as, MIP probe) (MIP-21Chr) and 1000 the specific oligonucleotide probe of the chromosome sequence for karyomit(e) 1 (such as, MIP probe) (MIP-1Chr) mixing.Ligase enzyme, polysaccharase and other reactive component are added in mixture.Sample hatches 4 minutes at 20 DEG C.Then sample is hatched 5 minutes to promote sex change at 95 DEG C, then 60 DEG C 15 minutes to promote the annealing of MIP probe and genomic dna.Then gap fill reaction is carried out with cyclisation MIP probe.(in some cases, end may not need gap fill react and directly connect).Nucleotide is added in sample, then hatch at 60 DEG C and be bonded to breach in MIP probe to make ligase enzyme and polysaccharase in 10 minutes.Then sample hatches 1 minute at 37 DEG C.Then, with exonuclease I and III processing sample thus digest remaining linear probe and ssDNA as not with the genomic dna of probe hybridization, 14 minutes are hatched to strengthen exonuclease activity subsequently in 37 DEG C, hatch 2 minutes with deactivation exonuclease in 95 DEG C, finally hatch 1 minute in 37 DEG C.Next uracil-N-glycosylase is added in sample, then hatches 10 minutes to promote enzymatic depurination at 37 DEG C, subsequently in 95 DEG C hatch 20 minutes with cut dealkalize base in MIP probe, the uracil residues of depurination.Now linearizing probe has inverted primer direction.
Next, droplet digital pcr is carried out to sample.Taq polysaccharase, universal primer, Taqman fluorescence probe and PCR reactive component are added in sample.By the Taqman fluorescence probe of the general probe binding sequence complementation on FAM dye marker and MIP-21Chr probe; By the Taqman fluorescence probe of the general probe binding sequence complementation on VIC dye marker and MIP-1Chr probe.Then be 100,000 monodispersed water-in-oil-type droplet by sample emulsification, stablize this droplet with surfactant additive, become emulsification oil phase and/or water-based PCR reacting phase.As a result, sample is assigned to 100, in 000 droplet.Then sample order about often drip each PCR reaction in droplet until terminal condition under experience 15-50 thermal cycling.Then droplet is analyzed by the transmitting of double-colored detection scheme detection FAM and VIC dyestuff.By determining that the mark of FAM fluorescent positive and negative droplet determines the target quantity counted for karyomit(e) 21.Similarly, by determining that the mark of VIC fluorescent positive and negative droplet determines the target quantity for counting with reference to sample (karyomit(e) 1).Then use the quantity of positive and negative droplet as the input value in Poisson's distribution, to determine in each droplet target karyomit(e) and with reference to chromosomal copy number (λ).Then with the Relative copy number of equation determination karyomit(e) 21 known in the art, such as, as Dube etc. (2008) PlosONE3 (8): described in e2876.doi:10.1371/journal.pone.0002876.Also by the degree of confidence of these equation determination estimated values.
Embodiment 4. is separated the positive in MIP reaction and negative droplet signal and ddPCR to the susceptibility of template copy numbers.
cyclization
The MIP cyclisation product of multiplex is prepared as follows: use 3-to weigh or the heavy probe library of 12-, its often kind of MIP comprised in every 10 μ L annealing mixtures in each 100 dusts mole (amol) multiple thing produces.1 dust mole equals 10
-18mole.100amol approximates ~ each MIP probe sequence of 60M copy.The volume of annealing reaction is 20ul.
(note, in this experiment, all volumes quoted in this programme are all double, start with 20ul annealing reaction; But all DNA, damping fluid and enzyme concn all remain identical in the 10ul annealing reaction scheme with standard).Probe library (IDTUltramers is prepared with the Macrodilution thing of selected MIP probe, by PAGE purifying), described MIP probe is selected from karyomit(e) 1 reference group (detecting with SEQIDNO:81) of 24 nucleic acid (SEQIDNO:1-24) or is selected from karyomit(e) 21 test group (detecting with SEQIDNO:82) of 24 nucleic acid (SEQIDNO:25-48).
The Raji people gDNA (0 of MIP probe and different copy number; 100; 1,000; Or 10,000 copy, 3pggDNA/ copies) mix in 1XAmpligase damping fluid in 96 hole PCR plate, in 95 DEG C of sex change 5 minutes in thermal cycler (EppendorfMastercyclerPro.S or ABI9700), then be cooled to 58 DEG C, and make it hatch and anneal > 12h in this temperature.
After annealing, be to maintain in thermal cycler while 58 DEG C, 0.75UAmpligase added in the 1XAmpligase damping fluid of 5 μ L in each reaction, mixing, to obtain the mixture that cumulative volume is 15 μ L, reseal flat board and hatch 15 minutes again at 58 DEG C.
the digestion of non-cyclisation material
After cyclization, 4 DEG C are down to immediately by slow for the temperature of thermal cycler, in each reacting hole, add the 5 μ L mixtures of 6UExoI and 30UExoIII in 1 × ExoIII damping fluid (EpiCentre) under mixing and reseal flat board (total reaction volume=20 μ L), exonuclease digestion is carried out to the excessive MIP probe of non-cyclisation and gDNA.Digestion reaction carries out 20 minutes in 37 DEG C in thermal cycler, subsequently 95 DEG C of sex change 10 minutes.
MIP reaction product is analyzed with qPCR (4 μ L cyclization mixture/20 μ LqPCR reaction solution), freezing at-20 DEG C subsequently, and store for droplet digital pcr (ddPCR) experiment.
general 2×
the preparation of stock solution
The general stock solution of following preparation (10mL)
General stock solution is stored in 4 DEG C, for multiple experiment.
the preparation of 2 × Hb_pr1ddPCR stock solution
Following preparation 2 × Hb_pr1ddPCR stock solution (520.5 μ L).
the preparation of 1.25 × Hb_pr1ddPCR stock solution
Following preparation 1.25 × HB_PR1ddPCR stock solution (800 μ L).
| Component | Volume (μ L) in every 800 μ L solution |
| 2x Hb_pr1 ddPCR stock solution | 520.5 |
| MgCl 2The aqueous solution (25mM) | 80 |
| Water | 199.5 |
| Cumulative volume | 800 |
1.25 × Hb_pr1ddPCR stock solution is assigned in 4 centrifuge tubes (1.5mL capacity) with 160 μ L aliquots.
the preparation of 2 × Hb_pr2ddPCR stock solution
Following preparation 2 × Hb_pr2ddPCR stock solution (936.9 μ L).
the preparation of 1.25 × Hb_pr2ddPCR stock solution
Following preparation 1.25 × Hb_pr2ddPCR stock solution (1440 μ L).
| Component | Volume (μ L) in every 1440 μ L solution |
| 2x Hb2 ddPCR stock solution | 936.9 |
| MgCl 2The aqueous solution (25mM) | 144 |
| Water | 359.1 |
| Cumulative volume | 1440 |
1.25 × Hb_pr2ddPCR stock solution is assigned in 8 centrifuge tubes (1.5mL capacity) with 160 μ L aliquots.
ddPCR program
The product of MIP cyclisation being tested melts and centrifugal (2,000rpm, 2min).By the MIP product of 40 μ L aliquots, namely from 2 × 20 μ L aliquots of duplicate determination reaction, combine with 160 μ L1.25 × Hb_pr1ddPCR stock solutions for MIP of design packet containing TaqmanAssayHb_pr1, or combine with the 1.25 × Hb2ddPCR stock solution for MIP of design packet containing TaqmanAssayHb_pr2.With the ChipShop droplet generation system with syringe pump system, reaction mixture is assigned as the droplet of 1nL.
Be transferred to by droplet sample (every droplet sample 3x30 μ L aliquot) on thermal cycler plate, be sealed envelope with paper tinsel, then thermal cycling is about 1.25h.By plate is started thermal cycling 94 DEG C of maintenances 10 minutes, make plate circulate through 35-40 circulation (94 DEG C, 20s/65 DEG C, 60s) subsequently, and finally cool and remain on 4 DEG C.The plate of thermal cycling stores at such a temperature.
By the video picture under Nikon opticmicroscope of remaining droplet aliquot, to evaluate homogeneity and suitable size.
The sample of thermal cycling is placed in QuantaLifeBox2Alpha detector system, wherein once from a hole, automatically draw droplet sample, and in single file by detector, this is for the fluorescence intensity evaluating droplet size and carry out self-reacting FAMTaqman probe.
According to their fluorescence amplitude, be positive or negative droplet by the droplet scoring in each hole of suitable size, and calculate the concentration of analyzed sample target according to Poisson statistics with these distributions.
Upper figure in Figure 10 shows, and the increase of positive droplet number (or counting) increases relevant to the input copy number of template DNA.Herein, this experiment uses Raji genomic dna (deriving from Raji cancer cells).For these experiments, use in the such as sample shown in first three columns (D4-6 in upper figure, the F4-6 in figure below) input copy be 0 copy (or without Template Controls " NTC ") DNA; In next group three row, (D7-9 in upper figure, the F7-9 in figure below) uses 100 copies; In next group three row, (E4-6 in upper figure, the G4-6 in figure below) uses 1000 copies; And for last three row, use 10,000 copy (E7-E9 in upper figure, the G7-G9 in figure below).In upper figure, use triple MIP and use three kinds of different MIP probes to carry out all MIP reactions, three kinds of different MIP probes go up different sites for test chromosome (i.e. karyomit(e) 21 also corresponds to hb_pr2) separately.For upper figure, left figure defines the threshold value between the positive and negative droplet at the vertical line at 10605RFU place on the sea line at 10605RFU (Relative fluorescence units) place and right figure.Carry out shown experiment in triplicate.
Figure below is the identical experiment of carrying out with one group of larger MIP probe.In figure below, use the heavy MIP of 12-, each wherein in 12 MIP probes is for the different zones in karyomit(e) 21.As shown in Figure 10, figure below display is for the positive droplet of about 4 times of quantity of the DNA profiling of given input number (such as, NTC, 100,1000,10000).Y-axle shows the Relative fluorescence units from the FAM signal (or Taqman probe) often dripping droplet emission.For upper figure and figure below, right figure provides the frequency histogram of the fluorescence amplitudes of the change of display droplet, and combines the data from all 12 hurdles shown in left figure.Herein, X-axle provides the Relative fluorescence units of FAM signal.For figure below, left figure defines the threshold value between the positive and negative droplet at the vertical line at 10,431RFU place on the sea line at 10,431RFU (Relative fluorescence units) place and right figure.
Figure 11 show, when MIP probe library from for reference to polynucleotide (hb_pr1 or karyomit(e) 1) probe time, the result of acquisition is similar to shown in Figure 10.The counting that this figure left frame goes out reflects the counting obtained with 3 heavy MIP probe libraries and 10,000 template DNA copied, and the counting that this figure right frame goes out reflects the counting obtained with 12 heavy MIP probe libraries and 10,000 template DNA copied.Carry out shown experiment in triplicate.
Figure 12 shows, and there is the template of 1000 copies or 10,000 copy in no matter reacting, hybridization efficiency is similar, and as working as template copy numbers by 1,000 increases to 10, counts shown by increase by 10 times when 000.Figure 12 also shows, and for the genomic dna of given copy number, can be increased the numerical value of counting by the multiplex degree increasing MIP probe.MIP probe allows at whole given karyomit(e) multiplex, provides a large amount of countings from a small amount of genome equivalent, and this is important for the little copy number change distinguished between target and reference.Carry out shown experiment in triplicate.
Although show and describe alternative embodiment of the present invention herein, to those skilled in the art clearly, these embodiments only provide by way of example.Without deviating from the invention, those skilled in the art will envision that a large amount of change, change and replacement.Should be appreciated that the various replacement schemes that can adopt embodiment of the present invention described herein in the embodiment of this invention.Claim is below intended to limit scope of the present invention, therefore contains the method and structure in these right and equivalent thereof.
Claims (53)
1. detect a method for the copy number of target polynucleotide in genetic material colony, comprising:
A. the first linking probe is combined with the first target polynucleotide;
B. the second linking probe is combined with the second target polynucleotide;
C. make described first linking probe and the second linking probe carry out ligation and be connected product to obtain one or more, 5 ' of described first linking probe end is connected to 3 ' end of described first linking probe and holds the 3 ' end being connected to described second linking probe by 5 ' of described second linking probe by wherein said ligation;
D. described one or more being connected product is assigned in two or more subregions;
E. the region in amplification one or more connection products described is to obtain amplified production;
F. determine the number of the described subregion comprising described amplified production, wherein in described determining step, described subregion keeps complete substantially; And
G. based on the Relative copy number of described first and second target polynucleotide of described number calculating of described subregion;
The direct object of wherein said method the diagnostic result of non-diseases.
2. the process of claim 1 wherein that described target polynucleotide is not assigned in two or more subregions described.
3. the process of claim 1 wherein during described amplification procedure, two or more subregions described keep complete substantially.
4. the process of claim 1 wherein that described first linking probe and the second linking probe are designed to separately in conjunction with described first target polynucleotide.
5. the process of claim 1 wherein that described allocation step (d) does not comprise and distribute described target polynucleotide.
6. the process of claim 1 wherein that two or more subregions described comprise from the initial amplified reaction of described connection product.
7. the process of claim 1 wherein that described first linking probe is designed to and plants between interior individuality conservative polynucleotide sequence and be combined.
8. the process of claim 1 wherein that described subregion is the water-based droplet be present in the mixture of at least two kinds of immiscible fluids.
9. the method for claim 8, wherein said water-based droplet is dispensed in oil-continuous phase.
10. the method for claim 1, comprises further and at least 4 linking probes is bonded to described first target polynucleotide.
11. the method for claim 1, comprise further and at least 4 linking probes are bonded to described first target polynucleotide, and at least 4 linking probes are bonded to described second target polynucleotide.
The method of 12. claims 1, wherein said first linking probe is designed in conjunction with the first area in described first target polynucleotide, described second linking probe is designed in conjunction with the second area in described first target polynucleotide, and wherein said first and second regions do not have identical sequence.
13. the process of claim 1 wherein that described first target polynucleotide is different from described second target polynucleotide.
The method of 14. claims 13, wherein said first target polynucleotide is test chromosome, and described second target polynucleotide is with reference to karyomit(e).
The method of 15. claims 14, wherein said test chromosome is selected from karyomit(e) 21, karyomit(e) 13, karyomit(e) 18 and X chromosome.
16. the process of claim 1 wherein described first target polynucleotide be selected from karyomit(e) 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, the karyomit(e) of X and Y.
17. the process of claim 1 wherein that described first target polynucleotide is chromosomal section.
The method of 18. claims 17, wherein said chromosomal section is relevant to fetus dysploidy.
19. the process of claim 1 wherein that the described 5 ' region of described first linking probe and described 3 ' region are designed to separately in conjunction with the flanking sequence in described first target polynucleotide.
20. the process of claim 1 wherein that the described 5 ' region of described first linking probe and described 3 ' region are designed to separately in conjunction with the contiguous sequence in described first target polynucleotide.
The method of 21. claims 20, wherein said contiguous sequence is separated by least 1 Nucleotide.
22. the method for claim 21, wherein said ligation comprises the gap fill reaction of template-driven further, to introduce Nucleotide in the region between the described 5 ' region and described 3 ' region of described first linking probe.
23. the process of claim 1 wherein that described first linking probe comprises the site that can digestedly cut.
The method of 24. claims 23, the wherein said site that can digestedly cut comprises one or more uridylic.
The method of 25. claims 23, the wherein said site that can digestedly cut comprises restriction site.
26. the process of claim 1 wherein that described first linking probe is snap close probe.
27. the process of claim 1 wherein that described first linking probe is molecular inversion probes.
The method of 28. claims 1, comprises further and carries out enzyme reaction to remove linear polynucleotides.
The method of 29. claims 1, comprises further and carries out enzyme reaction to remove single stranded polynucleotide.
30. the process of claim 1 wherein that described first linking probe is coupled to the first signal reagent, and wherein said second linking probe is coupled to second signal reagent.
The method of 31. claims 30, wherein said first signal reagent is the fluorescent marker of the first color, and described second signal reagent is the fluorescent marker of the second color.
32. the method for claim 31, wherein said determining step (f) comprises detecting and has described second linking probe that described first linking probe of the first signal reagent and detection have second signal reagent.
The method of 33. claims 32, wherein said first linking probe comprises many linking probes, wherein every bar probe is for the different zones of the first chromosome, and wherein said second linking probe comprises many linking probes, and wherein every bar probe is for the second chromosomal different zones.
34. the process of claim 1 wherein that described first target polynucleotide is test chromosome, and described second target polynucleotide is with reference to karyomit(e).
The method of 35. claims 30, wherein said first linking probe and the second linking probe are coupled to the signal reagent with identical signal colour.
36. 1 kinds of methods detecting the copy number of target polynucleotide in genetic material colony, comprising:
A. the first linking probe is combined with the first target polynucleotide;
B. the second linking probe is combined with the second target polynucleotide;
C. make described first and second linking probes carry out ligation and connect product to obtain one or more, 5 ' of described first linking probe end is connected to 3 ' end of described first linking probe and holds the 3 ' end being connected to described second linking probe by 5 ' of described second linking probe by wherein said ligation;
D. described one or more being connected products is assigned in two or more water-based droplets in oil-continuous phase;
E. the sequence in amplification one or more connection products described is to obtain amplified production;
F. determine the number of two or more water-based droplets described comprising described amplified production, wherein in described determining step, described water-based droplet keeps complete substantially; And
G. the Relative copy number of described first and second target polynucleotide is calculated;
The direct object of wherein said method the diagnostic result of non-diseases.
The method of 37. claims 36, wherein said target polynucleotide is not assigned in two or more water-based droplets described.
38. the method for claim 36, two or more water-based droplet average packets wherein said contain more than a linking probe, and described method comprises the mean number calculating target linking probe in each water-based droplet with algorithm further.
The method of 39. claims 36, two or more water-based droplets of wherein said step (d) more than 4,000 droplet.
40. the method for claim 39, wherein said more than 4,000 droplet is in one chamber with higher than 100, and the density combinations of 000 water-based droplet/ml together.
The method of 41. claims 36, wherein said droplet is single dispersing droplet.
42. the method for claim 36, wherein said method can be less than 1 comprising, described first target polynucleotide of detection in the genetic material colony of described first target polynucleotide of 000 copy.
43. the method for claim 36, two or more water-based droplet average packets wherein said contain more than a linking probe, and described method comprises the mean number calculating target linking probe in each water-based droplet with algorithm further.
The method of 44. claims 36, each in two or more water-based droplets wherein said on average has the diameter of 50nm-300 μm.
The method of 45. claims 36, wherein said first target polynucleotide is the chromosome segment relevant to genetic diseases.
The method of 46. claims 36, wherein said first linking probe is snap close probe.
The method of 47. claims 36, wherein said first linking probe is molecular inversion probes.
The method of 48. claims 36, two or more water-based droplets wherein said do not comprise microballon that is a large amount of and oligonucleotide coupling.
The method of 49. claims 36, wherein said oil-continuous phase comprises anionic fluorosurfactants.
The method of 50. claims 49, wherein said anionic fluorosurfactants is anionic fluorosurfactants Krytox
tMammonium salt.
The method of 51. claims 50, wherein said Krytox is selected from the morpholino derivative of KrytoxAS, KrytoxFSH and KrytoxFSH.
The method of 52. claims 36, wherein said oil phase comprises fluorinated oil.
The method of 53. claims 36, two or more water-based droplets wherein said do not comprise the microballon with oligonucleotide coupling.
Applications Claiming Priority (25)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US26459109P | 2009-11-25 | 2009-11-25 | |
| US61/264,591 | 2009-11-25 | ||
| US30984510P | 2010-03-02 | 2010-03-02 | |
| US30983710P | 2010-03-02 | 2010-03-02 | |
| US61/309,837 | 2010-03-02 | ||
| US61/309,845 | 2010-03-02 | ||
| US31763910P | 2010-03-25 | 2010-03-25 | |
| US31763510P | 2010-03-25 | 2010-03-25 | |
| US34121810P | 2010-03-25 | 2010-03-25 | |
| US31768410P | 2010-03-25 | 2010-03-25 | |
| US34106510P | 2010-03-25 | 2010-03-25 | |
| US61/317,639 | 2010-03-25 | ||
| US61/317,684 | 2010-03-25 | ||
| US61/317,635 | 2010-03-25 | ||
| US61/341,065 | 2010-03-25 | ||
| US61/341,218 | 2010-03-25 | ||
| US38098110P | 2010-09-08 | 2010-09-08 | |
| US61/380,981 | 2010-09-08 | ||
| US40910610P | 2010-11-01 | 2010-11-01 | |
| US61/409,106 | 2010-11-01 | ||
| US10947310P | 2010-11-02 | 2010-11-02 | |
| US61/109,473 | 2010-11-02 | ||
| US41076910P | 2010-11-05 | 2010-11-05 | |
| US61/410,769 | 2010-11-05 | ||
| PCT/US2010/058124 WO2011066476A1 (en) | 2009-11-25 | 2010-11-25 | Methods and compositions for detecting genetic material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102985552A CN102985552A (en) | 2013-03-20 |
| CN102985552B true CN102985552B (en) | 2016-02-17 |
Family
ID=44066935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201080062146.9A Active CN102985552B (en) | 2009-11-25 | 2010-11-25 | For detecting the method and composition of genetic material |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN102985552B (en) |
| CA (1) | CA2767028A1 (en) |
| GB (1) | GB2485850C (en) |
Families Citing this family (50)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10273541B2 (en) | 2012-08-14 | 2019-04-30 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US9701998B2 (en) | 2012-12-14 | 2017-07-11 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US10584381B2 (en) | 2012-08-14 | 2020-03-10 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US9951386B2 (en) | 2014-06-26 | 2018-04-24 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US10221442B2 (en) | 2012-08-14 | 2019-03-05 | 10X Genomics, Inc. | Compositions and methods for sample processing |
| US10323279B2 (en) | 2012-08-14 | 2019-06-18 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US11591637B2 (en) | 2012-08-14 | 2023-02-28 | 10X Genomics, Inc. | Compositions and methods for sample processing |
| US10752949B2 (en) | 2012-08-14 | 2020-08-25 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US9567631B2 (en) | 2012-12-14 | 2017-02-14 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| CN104769127A (en) | 2012-08-14 | 2015-07-08 | 10X基因组学有限公司 | Microcapsule compositions and methods |
| US10533221B2 (en) | 2012-12-14 | 2020-01-14 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| EP3862435A1 (en) * | 2013-02-08 | 2021-08-11 | 10X Genomics, Inc. | Polynucleotide barcode generation |
| EP2986762B1 (en) * | 2013-04-19 | 2019-11-06 | Bio-Rad Laboratories, Inc. | Digital analyte analysis |
| US9944998B2 (en) * | 2013-07-25 | 2018-04-17 | Bio-Rad Laboratories, Inc. | Genetic assays |
| US10395758B2 (en) | 2013-08-30 | 2019-08-27 | 10X Genomics, Inc. | Sequencing methods |
| WO2015035555A1 (en) * | 2013-09-10 | 2015-03-19 | 深圳华大基因科技有限公司 | Method, system, and computer readable medium for determining whether fetus has abnormal number of sex chromosomes |
| US9824068B2 (en) | 2013-12-16 | 2017-11-21 | 10X Genomics, Inc. | Methods and apparatus for sorting data |
| US9694361B2 (en) | 2014-04-10 | 2017-07-04 | 10X Genomics, Inc. | Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same |
| CA2952503A1 (en) | 2014-06-26 | 2015-12-30 | 10X Genomics, Inc. | Processes and systems for nucleic acid sequence assembly |
| CN113249435B (en) | 2014-06-26 | 2024-09-03 | 10X基因组学有限公司 | Method for analyzing nucleic acid from single cell or cell population |
| CN104032031A (en) * | 2014-07-04 | 2014-09-10 | 华东理工大学 | PCR analyzing method for quantitatively detecting nucleic acid through RNA polymerase and ligase coupled reaction medium |
| BR112017008877A2 (en) | 2014-10-29 | 2018-07-03 | 10X Genomics Inc | methods and compositions for targeting nucleic acid sequencing |
| US9975122B2 (en) | 2014-11-05 | 2018-05-22 | 10X Genomics, Inc. | Instrument systems for integrated sample processing |
| US10221436B2 (en) | 2015-01-12 | 2019-03-05 | 10X Genomics, Inc. | Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same |
| SG11201705425SA (en) | 2015-01-13 | 2017-08-30 | 10X Genomics Inc | Systems and methods for visualizing structural variation and phasing information |
| WO2016130578A1 (en) | 2015-02-09 | 2016-08-18 | 10X Genomics, Inc. | Systems and methods for determining structural variation and phasing using variant call data |
| EP4286516A3 (en) | 2015-02-24 | 2024-03-06 | 10X Genomics, Inc. | Partition processing methods and systems |
| KR20170119710A (en) | 2015-02-24 | 2017-10-27 | 10엑스 제노믹스, 인크. | Targeted nucleic acid sequence coverage method |
| CN105936930A (en) * | 2015-03-04 | 2016-09-14 | 松下知识产权经营株式会社 | DNA detection method and DNA detection device |
| CN105969655A (en) * | 2015-03-10 | 2016-09-28 | 松下知识产权经营株式会社 | Method for analyzing multiple nucleic acid targets |
| CN108350500A (en) * | 2015-07-29 | 2018-07-31 | 普罗格尼迪公司 | Nucleic acid for detecting chromosome abnormality and method |
| WO2017074094A1 (en) * | 2015-10-29 | 2017-05-04 | Biocore Co., Ltd. | A method for prenatal diagnosis using digital pcr. |
| KR101848438B1 (en) * | 2015-10-29 | 2018-04-13 | 바이오코아 주식회사 | A method for prenatal diagnosis using digital PCR |
| SG10202108763UA (en) | 2015-12-04 | 2021-09-29 | 10X Genomics Inc | Methods and compositions for nucleic acid analysis |
| JP6735348B2 (en) | 2016-02-11 | 2020-08-05 | １０エックス ジェノミクス， インコーポレイテッド | Systems, methods and media for de novo assembly of whole genome sequence data |
| CN105821482B (en) * | 2016-04-29 | 2018-04-10 | 李星军 | A kind of biochemistry micro- reaction system, high-flux sequence build storehouse instrument and application |
| WO2017197338A1 (en) | 2016-05-13 | 2017-11-16 | 10X Genomics, Inc. | Microfluidic systems and methods of use |
| US10011872B1 (en) | 2016-12-22 | 2018-07-03 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US10550429B2 (en) | 2016-12-22 | 2020-02-04 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| US10815525B2 (en) | 2016-12-22 | 2020-10-27 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
| EP3545089B1 (en) | 2017-01-30 | 2022-03-09 | 10X Genomics, Inc. | Methods and systems for droplet-based single cell barcoding |
| WO2018213774A1 (en) | 2017-05-19 | 2018-11-22 | 10X Genomics, Inc. | Systems and methods for analyzing datasets |
| EP3445876B1 (en) | 2017-05-26 | 2023-07-05 | 10X Genomics, Inc. | Single cell analysis of transposase accessible chromatin |
| US10400235B2 (en) | 2017-05-26 | 2019-09-03 | 10X Genomics, Inc. | Single cell analysis of transposase accessible chromatin |
| CN107619827B (en) * | 2017-09-12 | 2020-11-13 | 广州阳普医疗科技股份有限公司 | Composition, preparation method thereof and application thereof in field of preservation of free DNA (deoxyribonucleic acid) in whole blood |
| EP3625361A1 (en) | 2017-11-15 | 2020-03-25 | 10X Genomics, Inc. | Functionalized gel beads |
| US10829815B2 (en) | 2017-11-17 | 2020-11-10 | 10X Genomics, Inc. | Methods and systems for associating physical and genetic properties of biological particles |
| EP3775271A1 (en) | 2018-04-06 | 2021-02-17 | 10X Genomics, Inc. | Systems and methods for quality control in single cell processing |
| EP3781710A4 (en) * | 2018-04-17 | 2022-01-05 | Chromacode, Inc. | Methods and systems for multiplex analysis |
| CN116194591A (en) * | 2020-06-10 | 2023-05-30 | 博纳迪诊断学有限责任公司 | Sequence conversion reaction |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007087312A2 (en) * | 2006-01-23 | 2007-08-02 | Population Genetics Technologies Ltd. | Molecular counting |
| US8460866B2 (en) * | 2006-03-01 | 2013-06-11 | Keygene N.V. | High throughput sequence-based detection of SNPs using ligation assays |
| EP2021113A2 (en) * | 2006-05-11 | 2009-02-11 | Raindance Technologies, Inc. | Microfluidic devices |
| EP3424598B1 (en) * | 2006-06-14 | 2022-06-08 | Verinata Health, Inc. | Rare cell analysis using sample splitting and dna tags |
| CA3149293C (en) * | 2008-09-23 | 2023-09-12 | Bio-Rad Laboratories, Inc. | Droplet-based assay system |
| US9156010B2 (en) * | 2008-09-23 | 2015-10-13 | Bio-Rad Laboratories, Inc. | Droplet-based assay system |
-
2010
- 2010-11-25 GB GB1100787.9A patent/GB2485850C/en active Active
- 2010-11-25 CA CA2767028A patent/CA2767028A1/en not_active Abandoned
- 2010-11-25 CN CN201080062146.9A patent/CN102985552B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| GB2485850A (en) | 2012-05-30 |
| CA2767028A1 (en) | 2011-06-03 |
| GB2485850C (en) | 2019-01-23 |
| CN102985552A (en) | 2013-03-20 |
| GB2485850B (en) | 2014-01-29 |
| GB201100787D0 (en) | 2011-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102985552B (en) | For detecting the method and composition of genetic material | |
| EP2504448B1 (en) | Methods and compositions for detecting genetic material | |
| US11499181B2 (en) | Analysis of nucleic acids | |
| US8206926B2 (en) | Restriction endonuclease enhanced polymorphic sequence detection | |
| US8652780B2 (en) | Restriction endonuclease enhanced polymorphic sequence detection | |
| US20230340590A1 (en) | Method for verifying bioassay samples | |
| CN108220392A (en) | Enrichment and the method for determining target nucleotide sequences | |
| JP2014512826A (en) | Methods and compositions for nucleic acid analysis | |
| US20190249238A1 (en) | Integrated single cell sequencing | |
| WO2018170659A1 (en) | Methods and compositions for preparing sequencing libraries | |
| US20160362729A1 (en) | Methods and compositions for chromosome mapping | |
| US20220355292A1 (en) | Methods and compositions for detecting genetic material | |
| WO2024059528A2 (en) | Method for estimation of fetal fraction in cell-free dna from maternal sample |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |