CN106566880A - Hereditary hearing loss gene mutation detection kit - Google Patents
Hereditary hearing loss gene mutation detection kit Download PDFInfo
- Publication number
- CN106566880A CN106566880A CN201610942873.3A CN201610942873A CN106566880A CN 106566880 A CN106566880 A CN 106566880A CN 201610942873 A CN201610942873 A CN 201610942873A CN 106566880 A CN106566880 A CN 106566880A
- Authority
- CN
- China
- Prior art keywords
- mutation detection
- gene mutation
- hearing impairment
- hereditary hearing
- probe
- 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.)
- Pending
Links
- 208000016354 hearing loss disease Diseases 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 50
- 206010011878 Deafness Diseases 0.000 title claims abstract description 47
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 32
- 230000010370 hearing loss Effects 0.000 title abstract 4
- 231100000888 hearing loss Toxicity 0.000 title abstract 4
- 239000000523 sample Substances 0.000 claims abstract description 76
- 238000009396 hybridization Methods 0.000 claims abstract description 21
- 238000012408 PCR amplification Methods 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 9
- 102000003960 Ligases Human genes 0.000 claims abstract description 7
- 108090000364 Ligases Proteins 0.000 claims abstract description 7
- 239000013641 positive control Substances 0.000 claims abstract description 5
- 239000003085 diluting agent Substances 0.000 claims abstract description 4
- 239000013642 negative control Substances 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 38
- 238000005859 coupling reaction Methods 0.000 claims description 20
- 108020004414 DNA Proteins 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 15
- 231100000895 deafness Toxicity 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 10
- 230000007850 degeneration Effects 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- 102200115376 rs111033318 Human genes 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 102200115471 rs111033220 Human genes 0.000 claims description 5
- 102200115469 rs111033305 Human genes 0.000 claims description 5
- 102200115374 rs121908362 Human genes 0.000 claims description 5
- 102200115372 rs121908363 Human genes 0.000 claims description 5
- 102220063029 rs200455203 Human genes 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 4
- 102200115475 rs201562855 Human genes 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000005515 coenzyme Substances 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 239000003599 detergent Substances 0.000 claims description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims 4
- 239000000376 reactant Substances 0.000 claims 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims 2
- 101000942941 Arabidopsis thaliana DNA ligase 6 Proteins 0.000 claims 1
- 210000004381 amniotic fluid Anatomy 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000004069 differentiation Effects 0.000 claims 1
- 210000005069 ears Anatomy 0.000 claims 1
- 230000002093 peripheral effect Effects 0.000 claims 1
- 230000035772 mutation Effects 0.000 abstract description 19
- 238000007838 multiplex ligation-dependent probe amplification Methods 0.000 abstract description 12
- 238000005516 engineering process Methods 0.000 abstract description 11
- 238000005251 capillar electrophoresis Methods 0.000 abstract description 3
- 101150098072 20 gene Proteins 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 9
- 230000002068 genetic effect Effects 0.000 description 8
- 230000000869 mutational effect Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 6
- 238000013461 design Methods 0.000 description 5
- 238000003908 quality control method Methods 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 108700028369 Alleles Proteins 0.000 description 3
- 101000889136 Homo sapiens Gap junction beta-3 protein Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 206010011882 Deafness congenital Diseases 0.000 description 2
- 102100037156 Gap junction beta-2 protein Human genes 0.000 description 2
- 101000954092 Homo sapiens Gap junction beta-2 protein Proteins 0.000 description 2
- 108020005196 Mitochondrial DNA Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 102100035278 Pendrin Human genes 0.000 description 2
- 108091006507 SLC26A4 Proteins 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000009946 DNA mutation Effects 0.000 description 1
- 206010011891 Deafness neurosensory Diseases 0.000 description 1
- 102100039397 Gap junction beta-3 protein Human genes 0.000 description 1
- 101150034593 Gjb2 gene Proteins 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 206010048865 Hypoacusis Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 101150030803 SLC26A4 gene Proteins 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012072 newborn hearing screening Methods 0.000 description 1
- 201000006790 nonsyndromic deafness Diseases 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000003793 prenatal diagnosis Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 102220030676 rs11231865 Human genes 0.000 description 1
- 102200008493 rs28933385 Human genes 0.000 description 1
- 102220174146 rs772608978 Human genes 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
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
- C12Q1/6827—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
- C12Q1/6876—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)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention a mutation detection kit used for performing qualitative detection on 20 gene loci related to hearing loss in a genome DNA. The kit comprises a DNA diluent, a hybridization reaction liquid, a hearing loss probe mixed liquid A, a hearing loss probe mixed liquid B, a connecting reaction liquid A, ligase, a connecting reaction liquid B, polymerase, a PCR amplification primer mixed liquid, positive control, and negative control. The kit provided by the invention performs gene mutation detection mainly by combination of a multiplex ligation-dependent probe amplification (MLPA) technology and capillary electrophoresis, so that multiple mutation loci can be detected at the same time; wild type, homozygous mutation type and heterozygosis mutation type of 20 loci can be accurately determined by two tubes concurrently; and meanwhile, a primer probe is also designed, so that quite high accuracy and specificity are achieved.
Description
Technical field
The invention belongs to biological technical field, major embodiment be MLPA methods and sequencing combination technology application, especially
It is related to a kind of non-comprehensive hereditary hearing impairment gene polymorphism sites detection.
Background technology
According to developed country count, per 1000 neonates in, just have 1 Deaf and Hard of Hearing Talents.In China, audition speech is residual
Disease person accounts for the 24.16% of people with disability's sum, 30,000 deaf youngsters of annual new life up to more than 27,800,000.Research speculate 60% deaf morbidity with
Heredity is relevant, and has more than 120 deaf gene identified.Also therefore, increasing country is all carrying out neonate ear
Deaf gene screening work.As State Council in 2011 prints and distributes《Program for the Chinese Women's Development (2011-2020)》With《Chinese youngster
Virgin development outline (2011-2020)》Promulgation, deaf examination even more further becomes hygiene departments at different levels and medical institutions
Important process, so as to strive realizing that newborn hearing screening rate reaches more than 60% before the year two thousand twenty.In the implementation process of this plan
In, various deaf gene detection schemes continuously emerge, and are cooperated by hospital, and the form that government checks successively falls in different regions
Ground.According to statistics, deaf gene detection limit about more than 100 ten thousand annual at present, in the five-year the neonate of year detection limit 10,000,000 is realized
（60% examination rate）Mean annual 60% compound growth rate.The evolution of technology, the contrast of clinical manipulation, allow more hospitals to wish
The detection product to upgrading is sought in prestige, to realize detection sales volume, minimum testing cost, the highest recall rate of maximum, and
Most comprehensive subsequent analysis and state-of-the-art high throughput sequencing technologies are supported.
Reported according to pertinent literature and the academic conference of deafness project research team of country, about 21% deafness patient is carried
GJB2 gene mutation, 14.5% deafness patient carries SLC26A4 gene mutation, the deaf trouble for having 3.8% and 0.6% respectively in addition
Person is mutated with mitochondrial DNA A1555G and C1494T.This result discloses sending out for Chinese non-syndromic hearing loss patient common deafness mutation
Raw frequency, it is determined that GJB2, SLC26A4, mitochondrial gene 12SrRNA（A1555G and C1494T is mutated）It is to cause Chinese big portion
Three most commonly seen genes for dividing hereditary hearing impairment to occur, the hereditary hearing impairment of about 70-80% is individual by the prominent of these three genes
Change causes.In the case where substantial amounts of Clinical detection data are supported, we count the deaf gene mutation position of most high frequency in Chinese population
Point.On this basis, being classified as 20 deaf gene mutational sites of detection target spot not only can cover most of deaf base
Because of mutational site, while being also the pathogenic mutation site related to hereditary hearing impairment of academic circles at present accreditation, it is additionally added in addition
2 mutational sites on GJB3 genes.GJB3 genes are relevant with the high frequency phonosensitive nerve deafness day after tomorrow, are China native country clones
First genetic diseasess gene, mutation ratio is about 1.2% in deaf crowd.
In recent years, the country reports the gene diagnosis kit of congenital deafness gene or susceptibility deaf gene in succession
And its detection method, to meet the needs that extensive examination is carried out to deaf-related gene mutation, deaf gene DNA is detected at present
Method be based primarily upon chip technology, sequencing technologies and detection technique of fluorescence, there is complex operation, time-consuming, high cost and right
Operator require high not enough problem.Zhongde Meilian Biotech Co., Ltd. Wuxi applied for a kind of heritability in 2013
Deaf gene fluorescence detection reagent kit, the invention be it is a kind of can be while detecting four deaf-related genes, 17 hot spot mutations
Detection kit.Its main method for adopting is fluorescent labeling and ARMS-PCR, is detected using multiplex PCR system, the party
Method has quick, easy feature, but the reagent judges inaccurate for closing on mutational site, simultaneously because glimmering using four colors
Signal, in instrument demand difficulty is increased, and these features limit the popularity of its application.
Clinically gene DNA molecular level is concentrated mainly on to the detection of deaf gene at present, with very high special knowledge
Other ability.The present invention combines many detection advantages of MLPA methods and sequencing technologies, in GJB2, GJB3, SLC26A4 and mitochondrion
Some are selected on gene simultaneously in the pleomorphism site site of CHINESE REGION hot spot mutation, optimization operating system is constituted, with inspection
Survey bit number of points it is many, it is high detect, it is inexpensive, easy to operate the advantages of.
The content of the invention
The purpose of the present invention is：A kind of high flux, accuracy height, high specificity hereditary hearing impairment are provided using MLPA methods
Gene detecting kit.
The technical solution used in the present invention is：
MLPA technologies have extremely strong application potential and vast potential for future development.Ligase detection reaction is a kind of high specific base
Because of detection technique, it is based primarily upon nucleic acid specificity Hybridization principle, only two oligonucleotide probes of SNP site upstream and downstream with treat
When surveying no base space between DNA sequence complete complementary, and two probes, could occur in the presence of TaqDNA ligases
Coupled reaction, the fluorescence signal for scanning institute's band on probe by fluorescence detection device afterwards determines fragment length, realizes to SNP positions
The detection of point.Based on MLPA know-whies, 1 ~ 20 can be realized by designing the probe combined with fluorescent detection and analysis of different length
The SNP typings in individual site are required.Each MLPA probe includes two oligonucleotide fragments, and each probe includes one section of primer sequence
Row and one section of specific sequence.In MLPA reactions, two oligonucleotide fragments are all hybridized with target sequence, afterwards using company
Connect enzyme and connect two parts probe.Coupled reaction high special, only when two probes hybridize completely with target sequence, i.e., target sequence with
Two sections of probes could be connected into a complete single nucleic acid strands by probe-specific sequence complete complementary, ligase；, whereas if
Target sequence is not fully complementary with probe sequence, even if the difference of only one of which base, may result in hybridization not exclusively, makes connection anti-
Should carry out.After the completion of coupled reaction, the probe connected with a pair of universal primer amplifications, the amplified production of each probe
Length is all unique, and scope is in 130～480bp.Finally, by capillary electrophoresis separation amplified production, software analysis, draw
Conclusion.Only when coupled reaction is completed, the amplified peak that subsequent PCR is expanded and collected correspondent probe can be just carried out, if inspection
There is point mutation or disappearance, amplification mutation in the target sequence of survey, then the amplified peak of correspondent probe will be lacked, reduces or increased,
Therefore, exception or point mutation of the target sequence whether with the presence of copy number just can determine whether according to the change of amplified peak.
A kind of hereditary hearing impairment gene detecting kit and application, it is characterised in that：Include following 20 polymorphic positions
The probe of point:35delG, G1975C, GIVS15+5A, T2027A, 299-300delAT, 167delT, 176-191del16,
235delC, C538T, AIVS7-2G, C1229T, G1226A, A1174T, A2168G, C1494T, A1555G, C2162T,
G598A,
C281T, G547A.
Further, test kit can be included such as the probe of 20 pleomorphism sites in table 1 below：
The every point probe sequence of table 1
Above probe, each site includes four allele-specific non-marked probes, is respectively used to hybridization and produces open country
Raw type and saltant type genes of interest base sequence, in addition also one universal primer, for expanding target gene base sequence, obtains
Obtain a large amount of target gene sequences.In table 1, " W " numbering refers to allele hybridization probe wild type, and " M " numbering refers to that allele is miscellaneous
Probe saltant type, " F " numbering is handed over to refer to left hybrid probe, " R " numbering refers to right hybrid probe.
The probe of 20 described pleomorphism sites is divided into two groups.
First group：299_300delAT, C538T, C2162T, AIVS7-2G, G589A, GIVS15+5A, 167delT,
Second group of G1226A, G1975C, 235delC, A1555G：T2027A, C1229T, 176_191de116, G547A, A1174T,
C281T, 35delG, A2168G, C1494T.This have the advantage that the probability for avoiding non-specific amplification.
DNA diluents involved in the present invention include 10mMpH8.2Tris-HCl, 0.1mMEDTA.
Hybridization reaction solution includes 1.5M KCl, 300mM pH8.5Tris-HCl, 1mM EDTA.
Coupled reaction liquid A includes 0.2mM coenzyme NADs.
Coupled reaction liquid B includes 2.6mM MgCl2,5mM pH8.5Tris-HCl, 0.013% nonionic detergent.
Probe mixed liquor of the deaf probe mixed liquor A comprising 11 sites.
Probe mixed liquor of the deaf probe mixed liquid B comprising 9 sites.
Pcr amplification primer thing mixed liquor 2.5nmoldNTPs, 10pmolPCR universal amplification primers.
The plasmid mixture in 20 saltant type sites of positive control deafness.
The plasmid mixture of negative control 20 wild type sites of deafness.
Ligase refers to the Taq DNA ligases for being purchased from NEB, and this is a kind of heat stability enzyme and with high specific, because
For only in two oligonucleotide probes of upstream and downstream and no base sky between DNA sequence complete complementary to be measured, and two probes
During gap, coupled reaction could occur in the presence of Taq DNA ligases.
Polymerase refers to the Therminator DNA Polymerase for being purchased from NEB, is mainly used in PCR amplifications.
Present invention also offers the using method of mentioned reagent box, specific techniqueflow is：
The collection of sample and preservation.
The extraction of sample.
The dilution of sample.
DNA degeneration.
DNA and probe hybridization.
Probe coupled reaction.
Pcr amplification reaction.
Upper machine sequencing.
Interpretation of result.
Using MLPA technologies, unmutated and mutation probe is separately designed for same mutational site, according to design
Product length different instructions different probe.2 pipe PCR are carried out using same sample to expand simultaneously, can simultaneously detect 20 sites, and
Determine its saltant type and wild type.
Beneficial effects of the present invention：The invention provides a kind of detection site quantity is more, accuracy high, high specificity and letter
Just effective method.Using the nucleic acid specificity Hybridization principle of ligase, the method that 2 pipe PCR are expanded is carried out using same sample,
20 pleomorphism sites of deaf-related gene can be simultaneously detected, and determines its saltant type and wild type.Detection institute use range
Extensively, it is adaptable to adult or the etiological diagnosises of child patient, genetic counselling and prenatal diagnosis, the deafness of various unknown causes, bag
Congenital deafness and posteriority deafness crowd are included, audition is normal, but have the crowd of deaf household heredity factors, general population is recessiveness
Pathogenic genetic mutation Carriage examination.20 hereditary hearing impairment mutational sites can be simultaneously detected in 24 hours, by hair
Cons electrophoresis separates amplified production, carries out software analysis, it was therefore concluded that and then auxiliary diagnosis are carried out to deaf gene heritability.
Description of the drawings
Fig. 1 is MLPA method Cleaning Principles.
Fig. 2 is first group of negative controls（Wild type）, vertical coordinate represents peak height, and abscissa represents genetic fragment size,
Abscissa numeral represents the site of gene, and " 1 " represents 299_300delAT, and clip size is 109nt." 2 " represent C538T, piece
Duan great little is 122nt." 3 " represent C2162T, and clip size is 134nt." 4 " represent AIVS7-2G, and clip size is 147nt.
" 5 " represent G589A, and clip size is 158nt." 6 " represent GIVS15+5A, and clip size is 169nt." 7 " represent
167delT, clip size is 184nt." 8 " represent G1226A, and clip size is 199nt." 9 " represent G1975C, clip size
For 214nt." 10 " represent 235delC, and clip size is 219nt." 11 " represent A1555G, and clip size is 295nt.
Fig. 3 is first group of positive reference substance（Saltant type）, vertical coordinate represents peak height, and abscissa represents genetic fragment size,
Abscissa numeral represents the site of gene, and " 1 " represents 299_300delAT, and clip size is 103nt." 2 " represent C538T, piece
Duan great little is 116nt." 3 " represent C2162T, and clip size is 128nt." 4 " represent AIVS7-2G, and clip size is 141nt.
" 5 " represent G589A, and clip size is 152nt." 6 " represent GIVS15+5A, and clip size is 163nt." 7 " represent
167delT, clip size is 178nt." 8 " represent G1226A, and clip size is 193nt." 9 " represent G1975C, clip size
For 208nt." 10 " represent 235delC, and clip size is 223nt." 11 " represent A1555G, and clip size is 289nt.
Fig. 4 is second group of cloudy reference substance（Wild type）, vertical coordinate represents peak height, and abscissa represents genetic fragment size, horizontal
Coordinate digital represents the site of gene, and " 1 " represents T2027A, and clip size is 108nt." 2 " represent C1229T, and clip size is
121nt." 3 " represent 176_191de116, and clip size is 135nt." 4 " represent G547A, and clip size is 146nt." 5 " generation
Table A 1174T, clip size is 159nt." 6 " represent C281T, and clip size is 174nt." 7 " represent 35delG, clip size
For 189nt." 8 " represent A2168G, and clip size is 204nt." 9 " represent C1494T,
Clip size is 276nt.
Fig. 5 is second group of positive reference substance（Saltant type）, vertical coordinate represents peak height, and abscissa represents genetic fragment size,
Abscissa numeral represents the site of gene, and " 1 " represents T2027A, and clip size is 102nt." 2 " represent C1229T clip sizes
For 115nt." 3 " represent 176_191de116, and clip size is 127nt." 4 " represent G547A, and clip size is 140nt.“5”
A1174T is represented, clip size is 153nt." 6 " represent C281T, and clip size is 168nt." 7 " represent 35delG, and fragment is big
It is little for 183nt." 8 " represent A2168G, and clip size is 198nt." 9 " represent C1494T, and clip size is 270nt.
Fig. 6 is the fragment analysis collection of illustrative plates of first group of gained sample, and vertical coordinate represents peak height, and abscissa represents that genetic fragment is big
It is little.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but is not limited thereto.
The design of detection hereditary hearing impairment gene probe
Early stage of the present invention is combined by probe design principle with practical situation, designs a large amount of detection hereditary hearing impairment gene mutation
Related locus probe, then select that specificity is high, the hybridization probe that sensitivity is high by substantial amounts of testing sieve, final screening
Go out the probe of related DNA mutation best results, and the mutation in these sites is sickness rate highest crowd, reaches 80% or so.
The method and step of detection hereditary hearing impairment test kit：
The extraction of sample：Using the standard EDTA anticoagulant vacuum test tube of nontoxic sterilizing, at arm bending or the back of the hand, vein is adopted
Blood 1-5mL, screws lid, carries out to identify and keep blood taking tube uprightly to place and send control laboratory to detect.Carried using poba gene group DNA
Take test kit to be extracted.
The dilution of sample
Dilution DNA sample system
DNA degeneration
The PCR pipe of sample to be tested is placed in PCR amplification instrument, the program of according to the form below carries out DNA reaction of degeneration.
DNA denaturation programs
DNA and probe hybridization
According to number of samples, the ratio of according to the form below takes out each component, is placed in after mixing in a centrifuge tube, to each DNA degeneration
3 L are added in product.
Hybridization system
The PCR pipe for adding hybridization mixture is placed in PCR amplification instrument, the program of according to the form below carries out hybridization.
Hybridization program
Probe coupled reaction
According to number of samples, the ratio of according to the form below takes out each component, is placed in a centrifuge tube after piping and druming mixing, to each hybridization
32 L are added in product, keeps sample as on PCR during sample-adding, now every part of hybridization system cumulative volume is 40 L.
Coupled reaction system
The PCR pipe for adding coupled reaction mixture is placed in PCR amplification instrument, the program of according to the form below is attached reaction.
Coupled reaction program
Pcr amplification reaction
According to number of samples, the ratio of according to the form below takes out each component, is placed in after mixing in a centrifuge tube, to each coupled reaction
10 L are added in product, brief centrifugation after mixing.Now every part of PCR reaction systems cumulative volume is 50 L.
PCR reaction systems
The PCR pipe for adding PCR mixture, sample to be tested is placed in PCR amplification instrument, the program of according to the form below enters performing PCR amplification instead
Should.
PCR reacts thermocycling program
Upper machine testing：DNA fragmentation analysis is carried out using ABI3500Dx instruments, 9uL HI- are added in each hole of 96 orifice plates
DI,
Internal standard 0.3-0.7 L is added simultaneously, and according to loading table the L of pcr amplification product 0.7 of correspondence sample is added.By what is prepared
Hair
Model is with reference to genetic analyzer capillary electrophoresis operation instruction on cons electrophoresis, and 4 DEG C of 2min. are then after 95 DEG C of degeneration 3min
Upper model is loaded on into load sample frame with reference to genetic analyzer operation instruction.
Analysis of test results
Reaction automatically saves result after terminating, and the position for occurring peak to sample is analyzed, and according to software gene locis are carried out
Analysis, using MLPA technologies, unmutated and mutation probe is separately designed for same mutational site, according to the product of design
Thing length different instructions different probe.If the equal appearance of unmutated and mutant probe in the same site of same gene, is heterozygous mutant；
Only unmutated probe appearance, is wild type；Only mutant probe appearance, is homozygous mutation.
Quality Control is compareed：The judgement of male/female result selects respectively mutational site/wild site as positive control/the moon
Property control, should there is the signal of corresponding saltant type/wild type, and peak value is computed being determined effectively.（2）Blank is not contained
Corresponding site, should not produce signal, and without correspondence peak value, or peak value is extremely low can ignore.Two quality control standards meet simultaneously more than,
Experiment judges effectively carry out subsequent analysis, and otherwise experiment judges invalid.
For example：As shown in Figures 2 and 3, site 1（299_300delAT）Wild type and saltant type there is peak, then should
Site is heterozygous mutant；If peak occurs in the wild type in only site 1 and saltant type does not have peak, the site is wild type；If position
There is not peak in 1 wild type of point and peak occurs in saltant type, and the site is homozygous mutation.Such as Fig. 4 and Fig. 5, site 1（T2027A）Open country
There is peak in raw type and saltant type, then be changed to be a little heterozygous mutant；If there is peak in the wild type in only site 1 and saltant type does not have
Peak, then the site is wild type；If peak does not occur in the wild type of site 1 and peak occurs in saltant type, the site is homozygous mutation.More than
Site is all same.
Interpretation：Experimental result is being met on the premise of Quality Control result receives standard with reference to below with reference to value scope.Fragment point
After the completion of analysis, the peak signal that collection is obtained is carried out into Analysis of test results.DQ（Dosage Quotients）For same in experiment
The mutant peaks signal of one gene locis and the ratio of wildtype peaks signal.
Any one in each gene loci in wild type or saltant type must have peak signal.The result of quality-control product must be controlled
System within claimed range, otherwise experimental result do not meet tolerance interval be considered as it is invalid；Positive quality control result is in corresponding base
Because signal peak occurs in saltant type site, and DQ values need to be more than 1.5, otherwise illustrate the failure of an experiment, point out experiment hybridization or PCR amplifications
Failure；The gene mutation position of blank result is extremely low with wild type position no signal peak or peak value, and otherwise experiment is possible to
There is pollution.
It is first group of gene locis peak figure as shown in Fig. 6 samples, peak position and wild type and saltant type is gone out according to site
Basis for estimation understand that only AIVS7-2G sites peak occur in wild type and saltant type, so this site is prominent for heterozygosity
Become, and there is peak in wild type position in other sites, so being wild type.
The above is presently preferred embodiments of the present invention, is not limited to the present invention, all principles in the present invention
With any modification, equivalent or the improvement made within spirit etc., it is included within protection scope of the present invention.
Claims (24)
1. 20 hereditary hearing impairment gene mutation detection kits, it is characterised in that：Include following information：DNA diluents,
Hybridization reaction solution, 20 deaf gene sites, deaf probe mixed liquor A, deaf probe mixed liquid B, coupled reaction liquid A, connections
Enzyme, coupled reaction liquid B, polymerase, pcr amplification primer thing mixed liquor, positive control, negative control.
2. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described DNA is dilute
Liquid composition is released for Tris-HCl, EDTA.
3. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described hybridization is anti-
The liquid composition is answered to be：KCl、Tris–HCl、EDTA.
4. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that 20 described ears
Deaf gene locis are divided into two groups, first group：299_300delAT, C538T, C2162T, AIVS7-2G, G589A, GIVS15+5A,
167delT, G1226A, G1975C, 235delC, A1555G；Second group：T2027A, C1229T, 176_191de116, G547A,
A1174T, C281T, 35delG, A2168G, C1494T.
5. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described ligase
Refer to：It is purchased from NEB Taq DNA ligases（NEB）.
6. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described connection is anti-
Liquid A is answered to refer to：Coenzyme NAD.
7. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described connection is anti-
Liquid B is answered to refer to：MgCl2, Tris-HCl, nonionic detergent.
8. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described PCR expands
Increase mixture to refer to：FAM upstreams universal primer, sequence is：5'-FAM-GGGTTCCCTAAGGGTTGGA-3'；Downstream is general
Primer, sequence is：5'-GTGCCAGCAAGATCCAATCTAGA-3', dNTPs.
9. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described polymerase
Refer to：It is purchased from NEB Therminator archaeal dna polymerases.
10. hereditary hearing impairment gene mutation detection kit according to claim 1, it is characterised in that described deafness
Probe mixed liquor A is referred to:The probe mixed liquor in 11 sites.
11. hereditary hearing impairment gene mutation detection kits according to claim 1, it is characterised in that described deafness
Probe mixed liquid B is referred to:The probe mixed liquor in 9 sites.
12. hereditary hearing impairment gene mutation detection kits according to claim 1, it is characterised in that the described positive
Control is referred to：The plasmid mixture in deaf 20 saltant type sites.
13. hereditary hearing impairment gene mutation detection kits according to claim 1, it is characterised in that described feminine gender
Control is referred to：The plasmid mixture of deaf 20 wild type sites.
14. hereditary hearing impairment gene mutation detection kits according to claim 2, it is characterised in that described DNA is dilute
Release liquid
Concentration be：Tris–HCl：10mM pH 8.2；EDTA：0.1mM.
15. hereditary hearing impairment gene mutation detection kits according to claim 3, it is characterised in that described hybridization
The concentration of reactant liquor is：KCl：1.5M；TRIS-HCL：30 mM pH8.5；EDTA：1mM.
16. hereditary hearing impairment gene mutation detection kits according to claim 6, it is characterised in that described connection
The concentration of reactant liquor A is：Coenzyme NAD concentration：0.2mM.
17. hereditary hearing impairment gene mutation detection kits according to claim 7, it is characterised in that described connection
The concentration of reactant liquor B is：MgCl2 concentration：2.6mM；Tris-HCl concentration：5 mM, pH8.5；Nonionic detersive agent concentration：
0.013%。
18. hereditary hearing impairment gene mutation detection kits according to claim 8, it is characterised in that described PCR expands
Increase mixture concentration be：10pmol.
19. hereditary hearing impairment gene mutation detection kits according to claim 10, it is characterised in that described deafness
The concentration of probe mixed liquor A is：1-4fmol.
20. hereditary hearing impairment gene mutation detection kits according to claim 11, it is characterised in that described deafness
The concentration of probe mixed liquid B is：1-4fmol.
21. hereditary hearing impairment gene mutation detection kits according to claim 12, it is characterised in that the described positive
The concentration of control is：1-4fmol.
22. hereditary hearing impairment gene mutation detection kits according to claim 13, it is characterised in that described feminine gender
The concentration of control is：1-4fmol.
The using method of the 23. hereditary hearing impairment gene mutation detection kits according to any one of claim 1～22, bag
Include following steps：DNA dilutes（5.00µL）And degeneration（98 DEG C, 5min；25 DEG C, hold）Afterwards, hybridized（DNA is miscellaneous with probe
Hand over reaction system：The L of DNA diluents 5.00, the L of hybridization reaction solution 1.5, deafness mixed probe (A/B) 1.5 L after degeneration）；It is miscellaneous
Hand over reaction condition：（95 DEG C, 60s；60 DEG C, 16-20h）, coupled reaction is carried out after hybridization（Probe coupled reaction system：It is miscellaneous
Hand over mixture, dH after reaction2The L of O 25, the L of coupled reaction liquid A 3, the L of coupled reaction liquid B 3, the L of ligase 1）；Coupled reaction
Condition：（54 DEG C, 15min；98 DEG C, 5min；20 DEG C, hold）, the laggard performing PCR amplified reaction of coupled reaction（Pcr amplification reaction body
System：Pcr amplification primer thing mixed liquor 2.00 L, dH2The L of O 7.5, the L of polymerase 0.5；Pcr amplification reaction condition：（（95 DEG C/30s,
60 DEG C/30s, 72 DEG C/60s, 35cycles）, 72 DEG C, 20min, 1cycle, 15 DEG C, hold), carry out after pcr amplification reaction
Upper machine testing, using ABI3500Dx instruments signal-obtaining and differentiation are carried out, and being qualitatively judged according to the RST of amplification curve is
It is no to there is exception.
The using method of 24. hereditary hearing impairment gene detecting kits according to claim 23, it is characterised in that：It is described
Sample be to include various samples such as Chinese Fetus amniotic fluid, human peripheral sample, umbilical blood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610942873.3A CN106566880A (en) | 2016-11-02 | 2016-11-02 | Hereditary hearing loss gene mutation detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610942873.3A CN106566880A (en) | 2016-11-02 | 2016-11-02 | Hereditary hearing loss gene mutation detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106566880A true CN106566880A (en) | 2017-04-19 |
Family
ID=58534845
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610942873.3A Pending CN106566880A (en) | 2016-11-02 | 2016-11-02 | Hereditary hearing loss gene mutation detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106566880A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531599A (en) * | 2018-05-08 | 2018-09-14 | 杭州泰领生物技术有限公司 | A kind of kit and method of people HLA-B*5801 genotype |
| CN109504753A (en) * | 2017-09-13 | 2019-03-22 | 上海伯豪生物技术有限公司 | Hereditary hearing impairment related gene detection chip kit |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140088796A (en) * | 2013-01-03 | 2014-07-11 | 경북대학교 산학협력단 | Marker composition for diagnosis of hereditary hearing loss in Korean population |
| CN104480215A (en) * | 2014-12-30 | 2015-04-01 | 宁波有成生物医药科技有限公司 | Gene joint detection method and kit |
| CN104651516A (en) * | 2015-03-02 | 2015-05-27 | 苏州市立医院 | Novel chromosome microdeletion/microduplication syndrome detection system and kit |
| CN104694629A (en) * | 2015-02-02 | 2015-06-10 | 江苏佰龄全基因生物医学技术有限公司 | Kit for detecting hereditary deafness susceptibility gene mutation |
| CN216140441U (en) * | 2021-08-19 | 2022-03-29 | 哈尔滨星云生物信息技术开发有限公司 | Kit for gene detection |
| US20220111002A1 (en) * | 2019-05-21 | 2022-04-14 | University Of Miami | Treatment of sensorineural deafness |
-
2016
- 2016-11-02 CN CN201610942873.3A patent/CN106566880A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140088796A (en) * | 2013-01-03 | 2014-07-11 | 경북대학교 산학협력단 | Marker composition for diagnosis of hereditary hearing loss in Korean population |
| CN104480215A (en) * | 2014-12-30 | 2015-04-01 | 宁波有成生物医药科技有限公司 | Gene joint detection method and kit |
| CN104694629A (en) * | 2015-02-02 | 2015-06-10 | 江苏佰龄全基因生物医学技术有限公司 | Kit for detecting hereditary deafness susceptibility gene mutation |
| CN104651516A (en) * | 2015-03-02 | 2015-05-27 | 苏州市立医院 | Novel chromosome microdeletion/microduplication syndrome detection system and kit |
| US20220111002A1 (en) * | 2019-05-21 | 2022-04-14 | University Of Miami | Treatment of sensorineural deafness |
| CN216140441U (en) * | 2021-08-19 | 2022-03-29 | 哈尔滨星云生物信息技术开发有限公司 | Kit for gene detection |
Non-Patent Citations (4)
| Title |
|---|
| M H SONG等: "Clinical evaluation of DFN3 patients with deletions in the POU3F4 locus and detection of carrier female using MLPA", 《CLIN GENET》 * |
| 王小竹等: "运用多重连接探针检测DMD", 《医学研究杂志》 * |
| 王谦等: "MLPA和多重PCR法检测假肥大性肌营养不良患者DMD基因突变", 《广东医学》 * |
| 胡晓等: "多重连接依赖性探针扩增技术及其应用进展", 《现代生物医学进展》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504753A (en) * | 2017-09-13 | 2019-03-22 | 上海伯豪生物技术有限公司 | Hereditary hearing impairment related gene detection chip kit |
| CN109504753B (en) * | 2017-09-13 | 2022-08-16 | 上海伯豪生物技术有限公司 | Genetic deafness related gene detection chip kit |
| CN108531599A (en) * | 2018-05-08 | 2018-09-14 | 杭州泰领生物技术有限公司 | A kind of kit and method of people HLA-B*5801 genotype |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chiu et al. | Determination of RhD zygosity: comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach | |
| Gill et al. | An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA | |
| Konstantinidis et al. | Live births following Karyomapping of human blastocysts: experience from clinical application of the method | |
| Vialard et al. | Prenatal BACs‐on‐BeadsTM: the prospective experience of five prenatal diagnosis laboratories | |
| Vrettou et al. | Real‐time PCR for single‐cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis | |
| WO2024027569A1 (en) | Haplotype construction method independent of proband | |
| Yan et al. | Reliable detection of paternal SNPs within deletion breakpoints for non-invasive prenatal exclusion of homozygous α0-thalassemia in maternal plasma | |
| US20200407799A1 (en) | Determining linear and circular forms of circulating nucleic acids | |
| WO2023001212A1 (en) | Gene chip, molecular probe combination and kit for analyzing sheep milk production performance, and use | |
| Yin et al. | Identification of a de novo fetal variant in osteogenesis imperfecta by targeted sequencing-based noninvasive prenatal testing | |
| Breveglieri et al. | Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays | |
| CN106939334B (en) | Method for detecting fetal DNA content in plasma of pregnant woman | |
| Fiorentino | Molecular genetic analysis of single cells | |
| Lefferts et al. | Evaluation of the nanosphere verigene® system and the verigene® F5/F2/MTHFR nucleic acid tests | |
| CN106566880A (en) | Hereditary hearing loss gene mutation detection kit | |
| CN109321651A (en) | A kind of composition, kit, sample treatment and application detecting people CYP2D6 gene pleiomorphism | |
| CN109182493A (en) | The primer and kit and its detection method of people's 16p11.2 microdeletion syndrome detection | |
| CN111235261B (en) | Kit for detecting human platelet-specific antigen HPA 1-29 genotyping | |
| Lee et al. | Validation of fragile X screening in the newborn population using a fit-for-purpose FMR1 PCR assay system | |
| CN109295192A (en) | A kind of composition, kit, sample treatment and application detecting people MDR1 gene pleiomorphism | |
| WO2018186687A1 (en) | Method for determining nucleic acid quality of biological sample | |
| Williams et al. | Molecular diagnosis of human disease | |
| Bakhtiar et al. | Omics technologies for clinical diagnosis and gene therapy: medical applications in human genetics | |
| JP2008182993A (en) | Method, program and system for judging gene examination result | |
| CN111172248A (en) | General kit for verifying copy number variation based on fragment analysis technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170419 |
|
| WD01 | Invention patent application deemed withdrawn after publication |