CN103233072B - High-flux mythelation detection technology for DNA (deoxyribonucleic acid) of complete genome - Google Patents
High-flux mythelation detection technology for DNA (deoxyribonucleic acid) of complete genome Download PDFInfo
- Publication number
- CN103233072B CN103233072B CN201310163085.0A CN201310163085A CN103233072B CN 103233072 B CN103233072 B CN 103233072B CN 201310163085 A CN201310163085 A CN 201310163085A CN 103233072 B CN103233072 B CN 103233072B
- Authority
- CN
- China
- Prior art keywords
- dna
- seq
- flux
- site
- primer
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention aims at providing a high-flux mythelation detection method for the DNA (deoxyribonucleic acid) of a complete genome. The method comprises the following steps that: enzyme digestion is performed on the DNA of the complete genome by methylated modification dependent restriction enzyme FspEI, wherein methylated modification on CG site or CHG site in the genome can be identified by the FspEI; labels with an equal length and a core demethylated site are generated after the enzyme digestion; and the sequence information of the methylated sites in the range of the complete genome can be obtained by performing a high-flux sequencing technology on a label library. Via the method, accurate location for the methylated modification sites can be realized, and the sequencing expense can be effectively reduced by enriching the methylated sites of the genome via the enzyme digestion, and directly sequencing and quantifying the sequences of the methylated labels; the experimental flow only needs two days; and the method has the advantages of being high in flux, simple and convenient to operate, low in cost, good in reliability and the like, as well as is an excellent high-flux mythelation detection method for a complete genome, which is suitable for non-modal organisms.
Description
Technical field
The invention belongs to DNA methylation detection technique field, be specifically related to a kind of high-throughput complete genome DNA detection technique that methylates.
Background technology:
DNA methylation (DNA methylation) is that a kind of important epigenetics of eukaryotic gene group DNA is modified, at DNA methylation transferring enzyme (DNA methyltransferase, DNMT), under effect, the methyl group of S-adenosylmethionine (SAM) is covalently bound on the cytosine(Cyt) of DNA molecular and forms 5-methylcytosine (5-
mc) process.DNA methylation plays an important role maintaining in the biological procedureses such as the generation of higher organism normal cell function, genetic imprinting, fetal development, aging and human tumor.In invertebrates, genomic DNA methylation level, by the expression pattern of regulatory gene, participates in regulating the adaptive process of body to environment.Therefore, obtain the data that methylate in all cytosine(Cyt)s site in full genome range, significant for the Space-time speciality research of epigenetics.
Along with methylate research deepen continuously, had multiple methylation analysis method with meet difference methylate research needs.In the research for full gene scope DNA methylation, the means of detection are mainly the screening in the full genomic methylation site based on chip platform and the methylation profiles analysis based on high-flux sequence platform.Wherein chip technology is the testing tool of comparatively perfect maturation in the research that methylates of model animals, there is high coverage, convenient and swift, cost performance high, the full genomic methylation chip of the mankind Human Methylation HD 450 releasing as Illumina comprises 450,000 CpG sites, can cover the gene of all NCBI annotations.Operating process is simple, can carry out the methylate accurate detection in site of high-throughput.But for the relatively deficient non-model animals of genetic information, its chip involves great expense, and the handiness that the site that methylates is selected is not high, thereby is difficult to utilize existing chip platform non-model animals to be carried out to the research of the full genomic methylation of high-throughput.
Along with new-generation sequencing technology flux improves constantly and the reduction of cost, there are at present a lot of full genomic methylation detection methods based on two generations order-checking platform to be applied, include full genome bisulfite order-checking (whole genome bisulfite sequencing, and expression bisulfite order-checking (the reduced representation bisulfite sequencing simplifying BS-seq), RRBS), methylate DNA co-immunoprecipitation order-checking (methylated DNA immunoprecipitation sequencing, MeDIP-seq), methylate DNA enrichment is in conjunction with high-flux sequence (methylated DNA binding domain sequencing, and methylation sensitive restriction enzyme order-checking (methylation-sensitive Restriction Enzyme sequencing MBD-Seq), MRE-seq) etc.These technology have its relative merits and the scope of application, as the DNA methylation detection method based on the processing of sulfurous hydrohalogenic acid salt, although the gold standard detecting as DNA methylation, but complicated operation (processing of heavy sulfurous hydrohalogenic acid salt), the required cost that checks order is higher, is not suitable for the species that the genomes such as shellfish are larger.MeDIP-Seq is the site that methylates of thereby the region that methylates on the genome of specific antibody enrichment being carried out high-flux sequence and obtained full genome range.But a large amount of DNA of this Technology Need, and antibody is expensive.
Summary of the invention
The object of the present invention is to provide a kind of high-throughput complete genome DNA methylation detecting method, be a kind ofly applicable to non-model animals, low cost, simple and quick high-throughput full genomic methylation detection method, to make up the deficiencies in the prior art.
Complete genome DNA methylation detecting method of the present invention, comprises following step:
1) genomic dna is carried out to enzyme with restriction endonuclease FspEI enzyme and cut, obtain endonuclease bamhi;
2) two ends of endonuclease bamhi are connected respectively to top connection, as the binding site of amplimer;
3) endonuclease bamhi that connects top connection is carried out to first round pcr amplification with primer, thereby enrichment joint connects correct endonuclease bamhi;
4) first round pcr amplification product is carried out to second with primer after gel-purified and take turns pcr amplification, introduce Barcode and build sequencing library;
5) sequencing library checks order; Sequencing data analysis is obtained to full genomic methylation information.
Wherein, step 2) in joint, for joint slx1 and slx2, wherein form two nucleotide fragments of slx1, its sequence is respectively 5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3 ' (SEQ ID NO:1) and 3 '-CGAGAAGGCTAGANNNNN-5 ' (SEQ ID NO:2), and wherein N is any in base A, T, G, C;
Two nucleotide fragments that form slx2, its sequence is respectively 5 '-GTGACTGGAGTTCAGAC
GTGTGCTCTTCCGATCT-3 ' (SEQ ID NO:3) and 3 '-CGAGAAGGCTAGANNNNN-5 ' (SEQ ID NO:2).
Primer in described step 3), is Slx-Primer 1 and Slx-Primer 2, and its nucleotide sequence is respectively:
Slx-Primer1：5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTA
CACGAC-3’?（SEQ?ID?NO:4）；
Slx-Primer?2：5'-GTGACTGGAGTTCAGACGTGT-3’?（SEQ?ID?NO:5）；
Primer in described step 4) is Slx-Primer 1 and Slx-Index Primer, and its nucleotide sequence is respectively:
Slx-Primer1：5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTA
CACGAC-3’?（SEQ?ID?NO:4）；
Slx-Index?Primer：
5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT-3 ' (SEQ ID NO:6); Wherein N is any in base A, T, G, C.
Present method can realize the accurate location of the decorating site that methylates, and cuts enrichment genomic methylation site by enzyme, directly the sequence label that methylates is checked order and quantitatively, can effectively reduce order-checking expense.Experiment flow only needs two days, has the advantages such as flux is high, easy and simple to handle, with low cost, good reliability, is the full genomic methylation detection method of a kind of good high-throughput that is applicable to non-model animals.
Accompanying drawing explanation
Fig. 1: the flow process of full genomic methylation detection method of the present invention and principle schematic.
Embodiment:
The novel full genomic methylation that the present invention has developed based on high-flux sequence platform detects analytical technology MethylRAD-Seq(Methylation-dependent restriction-site associated DNA sequencing), modify the feature of dependency restriction endonuclease and high-flux sequence in conjunction with methyl, under the prerequisite that does not need genome background information, can excavate on a large scale the site that methylates in full genome range, directly accurately detect methylated cytosine(Cyt) site occurs.The principle of this technology is mainly to utilize the modification dependence limits enzyme FspEI that methylates to carry out enzyme to genomic dna to cut, methylating in genome on CG or CHG site modified and all can be identified by FspEI, enzyme is cut rear generation and is had the methylate isometric label in site of core, tag library is carried out to high throughput sequencing technologies and can obtain the site sequence information that methylates in full genome range.Build storehouse simple flow quick, can carry out complete genome DNA methylation analysis to multiple samples simultaneously.And the joint that the present invention is used and primer can operate genomic dna efficiently, have improved efficiency.Method of the present invention is the full genomic methylation analytical procedure of a kind of cost high-throughput lower, easy and simple to handle for the relatively less non-model animals of epigenetics background.
For noun related in the present invention, be defined as follows:
1, restriction endonuclease, is called again endonuclease (endonuclease) in hydrolase nucleic acid, is the enzyme of the inner phosphodiester bond generation of hydrolyzable molecular chain oligonucleotide; The present invention's FspEI restriction endonuclease used is that one methylates and modifies dependency restriction endonuclease, modifies DNA molecular is produced to cutting action thereby depend on methylating of cytosine(Cyt) site.This enzyme is purchased from NEB(New England Biolabs) company limited.
2, joint: adaptor DNA, be one section short containing restriction enzyme site the artificial-synthetic DNA's fragment that can mate with passivity end or sticky end, linker DNA is usually used in being connected of a passivity end DNA and a sticky end DNA.Sometimes the linker DNA that is connected to sticky end is in order to provide one section of known sequence to unknown DNA fragmentation, and according to its design primer, unknown DNA fragmentation increases.
3, wherein N is any in base A, T, G, C; Wherein four kinds of deoxynucleosides of A, T, G, C representative composition DNA molecular.
4, the i.e. one section of short characteristic sequence of Barcode, when multiple samples are carried out to high-flux sequence simultaneously, to every reads upper with one section of specific short sequence (being barcode) order-checking accurately recognition sample originate.
Method of the present invention, includes following step:
1) prepare biological genome DNA: extract biological genomic dna, 4 ℃ of Refrigerator stores are for subsequent use.
The genomic dna utilization of extraction is methylated and modifies dependency restriction endonuclease FspEI enzyme and cut genome, obtain the label that methylates in full genome range, wherein the enzyme system of cutting is 20 μ l, comprise 300ng genomic dna, the FspEI restriction endonuclease (NEB) of 4U, 1 × NE Buffer4,1 × Enzyme Activator Solution, 1 × BSA, 37 ℃ are incubated 4 hours.
2) be designed with the joint of sticky end, connect label
Label 5 ' end that endonuclease reaction produces is all outstanding with 4 bases, the joint Slx-Adaptor 1 of design 3 ' end with 4 merger bases, Slx-Adaptor 2, ligation system is 20 μ l, the enzyme that comprises step on 10 μ l is cut product, 800U T4 DNA ligase (NEB), 1 × T4 Ligase Buffer, 4 μ M Slex-Ad1,4 μ M Slx-Ad2,20mM adenosine triphosphate atp, 4 ℃ connect 16h.
The sequence information of its center tap is in table 1.
3) carry out first round pcr amplification, enrichment label, wherein PCR reaction system is 20 μ L, comprises endonuclease bamhi that 7 μ l have connected joint as reaction template, 4 μ M Slx-Primer1 primers
(5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'), 4 μ M Slx-Primer2 primers (5'-GTGACTGGAGTTCAGACGTGT-3'), 0.3mM dNTPs, the super fidelity dna polysaccharase of 0.4U Phusion (NEB), 1 × HF buffer; Reaction conditions is 98 ℃ of sex change 5 s, 60 ℃ of annealing 20 s, and 72 ℃ are extended 10 s, carry out 14-18 circulation, and last 72 ℃ are extended 5min.8% non-denaturing polyacrylamide fine jade detected through gel electrophoresis for PCR product, amplified production size is about 120bp, cuts glue and reclaims PCR product.
4) Barcode Auele Specific Primer two is taken turns pcr amplification
In order to realize multiple individual detections that methylate of checking order that mix, can, by distinguishing the different Barcode of each individual interpolation, utilize the different primers of PCR reaction to introduce different Barcode.PCR reaction system is 20 μ L, comprises 25ng mono-and takes turns pcr amplification purified product, and 4 μ M Slx-Primer1 primer 5 '-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3',
4 μ M Slx-Index Primer primers:
5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT-3', wherein NNNNNN can change according to different Barcode sequences), 0.3mM dNTPs, the super fidelity dna polysaccharase of 0.4U Phusion (NEB), 1 × HF buffer; Reaction conditions is 98 ℃ of sex change 5 s, 60 ℃ of annealing 20 s, and 72 ℃ are extended 10 s, carry out 5-7 circulation, and last 72 ℃ are extended 5min.Parallel amplification 3 is managed, and PCR product detects with 8% native polyacrylamide gel electrophoresis, and amplified production size is about 150bp, utilizes QIAGEN PCR product purification test kit to reclaim purified pcr product.Utilize the order-checking of Solexa Hiseq2000 order-checking platform, this part is completed by order-checking company.
5) data analysis:
The method that the present embodiment adopts is as follows:
1, the destination file that Illunima/Solexa order-checking produces is fastq form, first utilizes SolexaQA software package to carry out quality filtration to original series, removes the reads that the sequence that contains N and the mass value that is greater than 5 bases are less than 10;
2, utilize CD-HIT software to carry out cluster analysis to short sequence, obtain methylate tag class and this in sequencing library and represent the abundance information of label, the covering reads number in the site that methylates can be weighed the methylation level in this site;
3, utilize SOAP software by the sequence alignment genome reference sequences in the site that methylates, can obtain the genome source-information in this site.
The joint relating in table 1 the present invention and primer sequence table
Describe the present invention as an example of Patinopecten yessoensis example in detail by embodiment below, for the present invention's reagent used, those skilled in the art can, according to technical scheme of the present invention, select in available reagent, and is not limited only to the restriction of the specific embodiment of the invention.
1) extract scallop genomic dna
Get each 12 of the Patinopecten yessoensis of II wild population in age and extra large great Jin shellfish, approximately 0.1 gram of each individual closed shell flesh, adds 500ulSTE lysis buffer (NaCl:100mM; EDTA:1mM, pH=8.0; Tris-HCl, 10nM, pH=8.0), shred, then add the SDS of 50 μ l 10%, and 5 μ l Proteinase Ks (20mg/ml), 56 ℃ of water-bath digestion, to organizing the complete cracking of fragment, lysate clarification.Add the saturated phenol of equal-volume (250 μ l) and chloroform/primary isoamyl alcohol (24:1) (250 μ l), extracting 3 times, get supernatant liquor, add (500ul) extracting 1 time of equal-volume chloroform/primary isoamyl alcohol (24:1), get supernatant liquor, add 1/10 volume NaAc(3M, pH 5.2) (dehydrated alcohol is preserved in 50 l) He 2 times of μ volume-20 ℃, and (1000 μ l), slowly shake up;-20 ℃ of centrifugal 10min of precipitation 30min.12000rpm, nucleic acid will be deposited in the pipe end.(l) washing precipitation be dried to ethanol and all volatilize of 1000 μ, adds 100 μ l sterilized waters to 70% ethanol and a small amount of (1-2 μ is RNase A l), and 4 ℃ of Refrigerator stores are for subsequent use.
The digestion of scallop genomic dna
Utilization methylates and modifies dependency restriction endonuclease FspEI enzyme and cut genome, obtain the label that methylates in full genome range: the enzyme system of cutting is 20 μ l, comprise 300ng genomic dna, the FspEI restriction endonuclease (NEB) of 4U, 1 × NEBuffer4,1 × Enzyme Activator Solution, 1 × BSA, 37 ℃ are incubated 4 hours.
2) two ends of endonuclease bamhi are connected respectively to top connection, as the binding site of amplimer
Label 5 ' end that endonuclease reaction produces is all outstanding with 4 bases, the joint Slx-Ad1 of design 3 ' end with 4 merger bases, Slx-Ad2, ligation system is 20ul, the enzyme that comprises the upper step of 10ul is cut product, 800U T4 DNA ligase (NEB), 1 × T4 Ligase Buffer, 4uM Adaptor Isosorbide-5-Nitrae uM Adaptor 2,20mM adenosine triphosphate atp, 4 ℃ connect 16h.
3) endonuclease bamhi that connects top connection is carried out to first round pcr amplification with primer, thereby enrichment joint connects correct endonuclease bamhi;
PCR reaction system is 20 μ L, comprise 7ul reaction template, 4 μ M Slx-Primer1 primers (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'), 4 μ M Slx-Primer2 primers (5'-GTGACTGGAGTTCAGACGTGT-3'), 0.3mM dNTPs, the super fidelity dna polysaccharase of 0.4U Phusion (NEB), 1 × HF buffer; Reaction conditions is 98 ℃ of sex change 5 s, 60 ℃ of annealing 20 s, and 72 ℃ are extended 10 s, carry out 14-18 circulation, and last 72 ℃ are extended 5min.8% non-denaturing polyacrylamide fine jade detected through gel electrophoresis for PCR product, amplified production size is about 120bp, cuts glue and reclaims PCR product.
4) first round pcr amplification product is carried out to second with primer and take turns pcr amplification, introduce Barcode and build sequencing library;
In order to realize multiple individual detections that methylate of checking order that mix, can, by distinguishing the different Barcode of each individual interpolation, utilize the different primers of PCR reaction to introduce different Barcode.PCR reaction system is 20uL, comprises 25ng mono-and takes turns pcr amplification purified product, 4 μ M Slx-Primer1 primer 5 '-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3', 4 μ M Slx-Index Primer primers
5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT-3', wherein NNNNNN can change according to different Barcode sequences, 24 Patinopecten yessoensis Slx-Index Primer primer sequence that library uses that methylates is as shown in table 2,0.3mM dNTPs, the super fidelity dna polysaccharase of 0.4U Phusion (NEB), 1 × HF buffer; Reaction conditions is 98 ℃ of sex change 5 s, 60 ℃ of annealing 20 s, and 72 ℃ are extended 10 s, carry out 5-7 circulation, and last 72 ℃ are extended 5min.Parallel amplification 3 is managed, and PCR product detects with 8% native polyacrylamide gel electrophoresis, and amplified production size is about 150bp, utilizes QIAGEN PCR product purification test kit to reclaim purified pcr product.Utilize the order-checking of Solexa Hiseq2000 order-checking platform, this part is completed by order-checking company.
The Slx-Index Primer primer sequence table relating in table 2 the present invention
| 1 | |
| 2 | |
| 3 | CAAGCAGAAGACGGCATACGAGATACAGTGGTGACTGGAGTTCAGACGTGT |
| 4 | |
| 5 | CAAGCAGAAGACGGCATACGAGATATCACGGTGACTGGAGTTCAGACGTGT |
| 6 | CAAGCAGAAGACGGCATACGAGATATGTCAGTGACTGGAGTTCAGACGTGT |
| 7 | CAAGCAGAAGACGGCATACGAGATCACGATGTGACTGGAGTTCAGACGTGT |
| 8 | CAAGCAGAAGACGGCATACGAGATCATCGGGTGACTGGAGTTCAGACGTGT |
| 9 | CAAGCAGAAGACGGCATACGAGATCATGGCGTGACTGGAGTTCAGACGTGT |
| 10 | CAAGCAGAAGACGGCATACGAGATCCATCTGTGACTGGAGTTCAGACGTGT |
| 11 | CAAGCAGAAGACGGCATACGAGATCGACATGTGACTGGAGTTCAGACGTGT |
| 12 | CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCAGACGTGT |
| 13 | CAAGCAGAAGACGGCATACGAGATCGTGCAGTGACTGGAGTTCAGACGTGT |
| 14 | CAAGCAGAAGACGGCATACGAGATCTCAGAGTGACTGGAGTTCAGACGTGT |
| 15 | CAAGCAGAAGACGGCATACGAGATCTGTAGGTGACTGGAGTTCAGACGTGT |
| 16 | CAAGCAGAAGACGGCATACGAGATGACATCGTGACTGGAGTTCAGACGTGT |
| 17 | CAAGCAGAAGACGGCATACGAGATGCACTAGTGACTGGAGTTCAGACGTGT |
| 18 | CAAGCAGAAGACGGCATACGAGATGCTCCAGTGACTGGAGTTCAGACGTGT |
| 19 | CAAGCAGAAGACGGCATACGAGATGTAGAGGTGACTGGAGTTCAGACGTGT |
| 20 | CAAGCAGAAGACGGCATACGAGATGTCTAGGTGACTGGAGTTCAGACGTGT |
| 21 | CAAGCAGAAGACGGCATACGAGATTAGTCAGTGACTGGAGTTCAGACGTGT |
| 22 | CAAGCAGAAGACGGCATACGAGATTCGGCAGTGACTGGAGTTCAGACGTGT |
| 23 | CAAGCAGAAGACGGCATACGAGATTGAGTGGTGACTGGAGTTCAGACGTGT |
| 24 | CAAGCAGAAGACGGCATACGAGATTGTAGCGTGACTGGAGTTCAGACGTGT |
Note: runic represents barcode sequence
5) sequencing library utilizes Solexa Hiseq2000 order-checking platform to check order; Sequencing data analysis is obtained to full genomic methylation information.
1, the destination file that Illumina/Solexa order-checking produces is fastq form, first utilize the perl script of compiling to carry out quality filtration to the original series in 24 libraries, the sequence that removal contains N and be greater than the reads that the mass value of 5 bases is less than 10, the high quality reads number range obtaining in 24 libraries that methylate is 6,749,208-14,661,303, the per-cent that the high quality reads number in each storehouse accounts for original reads is all more than 99%.
2, utilize CD-HIT software to carry out cluster analysis to high quality sequence label, obtain methylate tag class and this in sequencing library and represent the abundance of label, i.e. the methylation level information in this site.In wild-type Patinopecten yessoensis and a kind of saltant type Patinopecten yessoensis new lines " extra large great Jin shellfish " genome range, obtain 98,754 sites that methylate, the annotation analysis of the site that methylates being compared on genome reference sequences and carry out gene shows, the gene with the site that methylates relates in various biological function and pathways metabolism, illustrates that the related pathways such as growth, breeding and immunity of shellfish may have the participation regulation and control of epigenetics mechanism.
3, utilize edgeR software to carry out methylation level variance analysis to the site of wild-type Patinopecten yessoensis and " extra large great Jin shellfish " full genome range, obtain methylate 2452, site of difference, in " extra large great Jin shellfish ", there are methylation level and the closed shell flesh carotenoid content in 8 sites to show stronger dependency, relation conefficient is more than 0.90, this type of site is compared in genome reference sequences, obtained and somely accumulated relevant candidate gene to carotenoid.In order to test the reliability of the technology of the present invention, design two groups of technology and repeated experiment, the poisson's coefficient of two groups of parallel controls all can reach more than 0.99.Verify the stable and reliability of the technology of the present invention.
Experimental results show that the technology of the present invention both can be for the screening in the site that methylates in full genome range, the variance analysis that also can modify for the site that methylates between different cells, tissue or sample, inquiring into DNA methylation affects the regulatory mechanism of genetic expression.
The present invention has the advantages that flux is high, efficiency is high, cost is low, is applicable to, in the full genome range analyzing and testing site that methylates, in the full genomic methylation of non-model animals detects, have good application potential.
Claims (1)
1. a complete genome DNA methylation detecting method, comprises following step:
1) genomic dna is carried out to enzyme with restriction endonuclease FspEI enzyme and cut, obtain endonuclease bamhi;
2) two ends of endonuclease bamhi are connected respectively to top connection, as the binding site of amplimer;
3) endonuclease bamhi that connects top connection is carried out to first round pcr amplification with primer, thereby enrichment joint connects correct endonuclease bamhi;
4) first round pcr amplification product is carried out to second with primer after gel-purified and take turns pcr amplification, introduce Barcode and build sequencing library;
5) sequencing library checks order; Sequencing data analysis is obtained to full genomic methylation information;
Wherein step 2) in joint, be joint slx1 and slx2, wherein form two nucleotide fragments of slx1, its sequence is respectively SEQ ID NO:1 and SEQ ID NO:2; Two nucleotide fragments that form slx2, its sequence is respectively SEQ ID NO:3 and SEQ ID NO:2;
Described step 3) in the nucleotide sequence of primer be respectively SEQ ID NO:4 and SEQ ID NO:5;
Described step 4) in the nucleotide sequence of primer be respectively SEQ ID NO:4 and SEQ ID NO:6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310163085.0A CN103233072B (en) | 2013-05-06 | 2013-05-06 | High-flux mythelation detection technology for DNA (deoxyribonucleic acid) of complete genome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310163085.0A CN103233072B (en) | 2013-05-06 | 2013-05-06 | High-flux mythelation detection technology for DNA (deoxyribonucleic acid) of complete genome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103233072A CN103233072A (en) | 2013-08-07 |
| CN103233072B true CN103233072B (en) | 2014-07-02 |
Family
ID=48881136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310163085.0A Expired - Fee Related CN103233072B (en) | 2013-05-06 | 2013-05-06 | High-flux mythelation detection technology for DNA (deoxyribonucleic acid) of complete genome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103233072B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555846B (en) * | 2013-11-07 | 2015-12-30 | 中国海洋大学 | A kind of high-throughput low cost S NP classifying method based on liquid phase molecule Hybridization principle |
| AU2015336938B2 (en) * | 2014-10-20 | 2022-01-27 | Commonwealth Scientific And Industrial Research Organisation | Genome methylation analysis |
| CN104357573B (en) * | 2014-11-19 | 2016-09-28 | 苏州圣苏新药开发有限公司 | The method of precise verification unknown gene methylation |
| CN104561294B (en) * | 2014-12-26 | 2018-03-30 | 北京诺禾致源科技股份有限公司 | The construction method and sequence measurement of Genotyping sequencing library |
| CN105986030A (en) * | 2016-02-03 | 2016-10-05 | 广州市基准医疗有限责任公司 | Methylated DNA detection method |
| CN106192021B (en) * | 2016-08-02 | 2017-04-26 | 中国海洋大学 | Method for constructing series connection RAD [restriction-site-associated DNA (deoxyribonucleic acid)] tag sequencing libraries |
| CN108319984B (en) * | 2018-02-06 | 2019-07-02 | 北京林业大学 | The construction method and prediction technique of xylophyta leaf morphology feature and photosynthesis characteristics prediction model based on DNA methylation level |
| CN109136363A (en) * | 2018-08-31 | 2019-01-04 | 领航基因科技（杭州）有限公司 | A method of the non-integral multiple kit of detection fetal chromosomal and detection fetal methylation DNA copy number |
| CN113166809B (en) * | 2018-12-29 | 2023-12-26 | 深圳华大生命科学研究院 | DNA methylation detection method, kit, device and application |
| CN111627499B (en) * | 2020-05-27 | 2020-12-08 | 广州市基准医疗有限责任公司 | Methylation level vectorization representation and specific sequencing interval detection method and device |
| CN113584600A (en) * | 2021-08-11 | 2021-11-02 | 翌圣生物科技(上海)股份有限公司 | Whole genome methylation single-stranded DNA library building method |
| CN115831367A (en) * | 2022-12-21 | 2023-03-21 | 北京博奥医学检验所有限公司 | Pregnancy complication risk prediction model and application thereof |
| CN116515955B (en) * | 2023-06-20 | 2023-11-17 | 中国科学院海洋研究所 | Multi-gene targeting typing method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CZ291877B6 (en) * | 1991-09-24 | 2003-06-18 | Keygene N.V. | Amplification method of at least one restriction fragment from a starting DNA and process for preparing an assembly of the amplified restriction fragments |
| EP2176428B1 (en) * | 2007-07-30 | 2017-04-12 | Roche Diagnostics GmbH | Methods of detecting methylated dna at a specific locus |
| CN101503733A (en) * | 2008-12-17 | 2009-08-12 | 上海人类基因组研究中心 | DNA cohesive end joint facilitating high throughput gene sequence label sequencing and use |
-
2013
- 2013-05-06 CN CN201310163085.0A patent/CN103233072B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103233072A (en) | 2013-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103233072B (en) | High-flux mythelation detection technology for DNA (deoxyribonucleic acid) of complete genome | |
| AU2018266377B2 (en) | Universal short adapters for indexing of polynucleotide samples | |
| JP7300989B2 (en) | Methods and systems for analyzing nucleic acid molecules | |
| Garrett-Bakelman et al. | Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution | |
| CN106192021B (en) | Method for constructing series connection RAD [restriction-site-associated DNA (deoxyribonucleic acid)] tag sequencing libraries | |
| Robinson et al. | Evaluation of affinity-based genome-wide DNA methylation data: effects of CpG density, amplification bias, and copy number variation | |
| WO2020160414A1 (en) | Compositions and methods for isolating cell-free dna | |
| Wolf et al. | The evolution of chloroplast genes and genomes in ferns | |
| Lv et al. | Advances in mapping the epigenetic modifications of 5‐methylcytosine (5mC), N6‐methyladenine (6mA), and N4‐methylcytosine (4mC) | |
| CN112752853A (en) | Determination of base modifications of nucleic acids | |
| Sucher et al. | DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants | |
| US20110237444A1 (en) | Methods of mapping genomic methylation patterns | |
| Good | Reduced representation methods for subgenomic enrichment and next-generation sequencing | |
| CN110546272A (en) | Method of attaching adapters to sample nucleic acids | |
| CN109880890B (en) | Detection method of goat HIAT1 gene insertion/deletion polymorphism and application thereof | |
| Larson et al. | A clinician’s guide to bioinformatics for next-generation sequencing | |
| US20200216885A1 (en) | Nucleic acid quantification products and processes | |
| WO2022199242A1 (en) | Set of barcode linkers and medium-flux multi-single-cell representative dna methylation library construction and sequencing method | |
| WO2011063210A2 (en) | Methods of mapping genomic methylation patterns | |
| Lyu et al. | Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists | |
| CN105506084B (en) | The rapidly and efficiently methylolated method of detection genomic DNA and kit | |
| US20200131566A1 (en) | Methods, compositions and systems for calibrating epigenetic partitioning assays | |
| EP4172357B1 (en) | Methods and compositions for analyzing nucleic acid | |
| JPWO2021067484A5 (en) | ||
| WO2021222828A1 (en) | Methods for sequence determination using partitioned nucleic acids |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140702 Termination date: 20150506 |
|
| EXPY | Termination of patent right or utility model |