CN105219851B - A kind of burnt sequencing quantitatively detects the method to methylate - Google Patents
A kind of burnt sequencing quantitatively detects the method to methylate Download PDFInfo
- Publication number
- CN105219851B CN105219851B CN201510621381.XA CN201510621381A CN105219851B CN 105219851 B CN105219851 B CN 105219851B CN 201510621381 A CN201510621381 A CN 201510621381A CN 105219851 B CN105219851 B CN 105219851B
- Authority
- CN
- China
- Prior art keywords
- sequencing
- nucleotide
- dgtp
- sequence
- dna
- 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
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 80
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 38
- 239000002773 nucleotide Substances 0.000 claims abstract description 35
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 35
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims abstract description 19
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 18
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 18
- 239000002253 acid Substances 0.000 claims abstract description 8
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 241000218636 Thuja Species 0.000 claims abstract description 3
- 239000002131 composite material Substances 0.000 claims abstract description 3
- 108020004414 DNA Proteins 0.000 claims description 44
- 238000004458 analytical method Methods 0.000 claims description 14
- 102000053602 DNA Human genes 0.000 claims description 12
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 12
- 238000012408 PCR amplification Methods 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 230000004048 modification Effects 0.000 claims description 9
- 238000012986 modification Methods 0.000 claims description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 6
- 108010090804 Streptavidin Proteins 0.000 claims description 5
- 238000012098 association analyses Methods 0.000 claims description 5
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 5
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 5
- 238000009396 hybridization Methods 0.000 claims description 5
- 230000004044 response Effects 0.000 claims description 5
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical group NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 239000000571 coke Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 108091028664 Ribonucleotide Proteins 0.000 claims description 2
- 238000013459 approach Methods 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 239000002336 ribonucleotide Substances 0.000 claims description 2
- 125000002652 ribonucleotide group Chemical group 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 230000011987 methylation Effects 0.000 abstract description 18
- 238000007069 methylation reaction Methods 0.000 abstract description 18
- 238000004445 quantitative analysis Methods 0.000 abstract description 5
- 230000008859 change Effects 0.000 abstract description 3
- 238000012175 pyrosequencing Methods 0.000 description 7
- 108091029430 CpG site Proteins 0.000 description 6
- 238000001712 DNA sequencing Methods 0.000 description 6
- 108091029523 CpG island Proteins 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229940079827 sodium hydrogen sulfite Drugs 0.000 description 3
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 3
- 101150028074 2 gene Proteins 0.000 description 2
- 102000007347 Apyrase Human genes 0.000 description 2
- 108010007730 Apyrase Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- 230000007067 DNA methylation Effects 0.000 description 2
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102000004523 Sulfate Adenylyltransferase Human genes 0.000 description 2
- 108010022348 Sulfate adenylyltransferase Proteins 0.000 description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical class OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- BEGBSFPALGFMJI-UHFFFAOYSA-N ethene;sodium Chemical group [Na].C=C BEGBSFPALGFMJI-UHFFFAOYSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 230000011218 segmentation Effects 0.000 description 2
- 238000005406 washing Methods 0.000 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 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 206010073069 Hepatic cancer Diseases 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 101710183568 Serine/threonine-protein kinase PknK Proteins 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000002790 cross-validation Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 101150051821 era gene Proteins 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000005257 nucleotidylation Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 125000003396 thiol group Chemical class [H]S* 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229940035893 uracil Drugs 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
- C12Q1/6869—Methods for sequencing
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The method to methylate is quantitatively detected the invention discloses a kind of burnt sequencing.This method is by PCR product of the genomic DNA after bisulfite conversion according to specific two nucleotide quickly through " A ", " G ", " T " region in DNA profiling sequence, and dGTP adds in " C " the content mode in DNA profiling sequence that measures and carries out burnt sequencing, continuously records each sequencing reaction and obtains information：It obtains dinuclear thuja acid composite number purpose coding and dGTP synthesizes information of number, by set in fixed target area sequence base C in all GC sites it is different degrees of change into T, dGTP synthesis information of number is recycled, realizes the quantitative analysis of each methylation sites in the sequence of target area.The present invention can increase substantially sequencing length, widened the analyst coverage of traditional burnt sequencing, be suitble to the quantitative analysis that methylates of list sample PCR product and mixing sample PCR product sequence.
Description
Technical field
The invention belongs to biological technical fields, are a kind of PCR product sequence analysis methods, and in particular to a kind of specific nucleosides
The burnt sequencing of synthesis that acid adds in quantitatively detects the method to methylate.
Background technology
The Human Genome Project and the development and completion of various Model organism genome plans, make the mankind step into rear gene
Epoch generate tremendous influence to the biological study and medical research in the present age, and molecular biology related discipline has obtained fast
Violent development.The difference of life, disease generation, the rule of development and the phase of drug and life entity are recognized from gene level
Interaction will be possibly realized.For gene sequencing, the emphasis of rear era gene is measured by whole genome sequence to be shifted
To the comparison to individual inheritance difference and object inter-species heritable difference in genome.More and more evidences show DNA methylation
Take part in the processes such as embryonic development, gene imprinting.Meanwhile it also plays very important effect in disease development.It methylates
The change of state is considered as to cause a key factor of cancer, this to change the drop for including genome entirety methylation level
The abnormal rise of Di He CpG islands part methylation level, so as to cause genome it is unstable (such as chromosome it is unstable, former
The expression of oncogene) and tumor suppressor gene do not express.
DNA methylation analysis includes quantification and qualification, and presently, there are many methods to select.Genomic DNA passes through
After crossing bisulf iotate-treated, cytimidine (C) residue not being methylated in original series is converted into uracil (U), and in PCR
Occur in amplification with thymidine (T)；And the cytimidine being methylated then remains unchanged.From this trunk of bisulfite conversion
Road sets out, afterwards can be with selectional restriction restriction analysis, real-time quantitative PCR, Sanger sequencings, pyrosequencing and high pass
Measure DNA sequencing etc..However, and not all method the quantitative data in single CpG sites can be provided.Sample through bisulfites at
The Sanger sequencings of PCR product had been considered as once the goldstandard in methylation analysis after reason, but if being surveyed to 5-10 clone
Sequence, this method are sxemiquantitative at most.High-throughput DNA sequencing brings very sensitive DNA first for the research that methylates
Base collection of illustrative plates covers entire CpG islands with single-molecule resolution.High-throughput DNA technique realizes more comprehensively covering for entire CpG islands
Lid, and single-molecule resolution provides unprecedented information for the heterogeneity of methylation patterns.It can parallel study it is multiple
Site.However, these advantages are also with some inferior positions, for example, a sizable investment, expensive reagents, turnaround time it is long,
Data analysis requirement is high.These features cause high-throughput DNA sequencing technology to be more suitable in genome range region of interest within
Determine, and to different samples the CpG sites of target area quantitative analysis due to sample size it is big, if each sample
It is analyzed using high-throughput DNA sequencing technology, then cost will be excessively huge.
Traditional pyrosequencing techniques can rapidly detect the frequency to methylate, to the methylation sites in sample into
Capable qualitative and quantitative detection.PCR product of the sample after bisulf iotate-treated is during coke sequencing extension, according to C and T
Incorporation quantitatively determine the C-T ratios of single locus.Therefore, the variation that methylates of different loci just can be detected accurately.
Since pyrosequencing provides real sequence data, methylation state is also just presented with sequence form.Hanoverian, Germany is cured
The research group of institute Anna Potapova leaders has carried out the cross validation of system to high-throughput DNA sequencing and burnt sequencing
(BMC Biotechnology,2011,11:6), to the methylation patterns in 12 sites in 10 primary carcinoma of liver samples in height
Statistical of the methylation level in the single CpG sites that flux DNA sequencing is obtained compared with traditional burnt sequencing analog value
Analysis shows that the methylation level in all single CpG sites suffers from splendid uniformity.It is one to show traditional Pyrosequencing
Simple, cheap analytical technology of the kind available for the CpG sites quantitative analysis of target area.
However, traditional Pyrosequencing needs the dNTP of addition respective volume due to each reaction, in this way, as sequencing is anti-
The progress answered, the volume of reactant is increasing, and the concentration of reactant is also just fewer and fewer, and the accuracy of sequencing is also more next
It is lower.Traditional burnt sequencing generally can only also measure the fragment length of about 60bp on the premise of nucleotide addition sequence is optimized
(Mashayekhi F, Ronaghi M.A.Analytical biochemistry, 2007,363,275-28719), and it is this
Each methylation sites need traditional Pyrosequencing of the realization of two sequencing reactions to the analysis of the PCR product that methylates just more
Add and limited be subject to sequencing length.A kind of method of accommodation is that this segment length's sequence is carried out burnt survey with the segmentation of multiple sequencing primers
Sequence.However, on the one hand, it can also increase analysis cost using multiple sequencing primers；On the other hand, due to by bisulfites
The cytimidine being methylated after reason then remains unchanged, therefore the degree that each site is methylated is different, two kinds in PCR product
The content of DNA profiling is also just different, causes the complexity of DNA profiling type, general is difficult to design multiple sequencing primers by its sequence
Segmentation is measured.The thus burnt sequencing Shortcomings in the quantitative detection to methylate to long segment sequence of tradition.Analysis conventional
Pyrosequencing is used to methylate quantitative detection it can be found that only make use of CpG sites (i.e. dGTP sequencings) information in sequencing, and
The sequencing information in other regions is simultaneously not used, and therefore, proposes that one kind synthesizes survey in real time based on two nucleotide according to our laboratories
Method (Xiao Pengfeng etc., the Chinese invention patent of sequence：ZL 2,012 1 0128597.6), by the sequencing in other regions with specific two
The single nucleotide synthesis order-checking of nucleotide substitution will undoubtedly reduce the number of synthesis order-checking, so as to which the reading for increasing sequencing is grown
Degree.
The content of the invention
In order to overcome the deficiencies in the prior art, the present invention provides a kind of burnt sequencing and quantitatively detects the side to methylate
The PCR product that method is sample after bisulf iotate-treated provide it is a kind of quick, efficiently, sensitively methylate quantitative analysis
Method.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of burnt sequencing quantitatively detects the method to methylate, and step includes：1) DNA is extracted；2) bisulf iotate-treated；3)
PCR amplification；4) it is sequenced；5) association analysis；
The sequencing approach of the step 4) is：Burnt sequencing is carried out according to specific two nucleotide, dGTP feed postitions；Wherein,
Two nucleotide measure serial response obtains composite number purpose coding information, dGTP measures serial response and obtains synthesis information of number.
The specific method of the step 4) comprises the following steps：
4-1) prepare single-stranded DNA templates：The magnetic bead of pcr amplification product and Streptavidin package is reacted, biotin is repaiied
The DNA chain of decorations is fixed on the magnetic bead, is denatured under 0.05-0.2M NaOH solutions；Loose another DNA chain is clear
It removes；Then with wash liquid, the single-stranded DNA templates being fixed；
4-2) sequencing primer hybridizes：By sequencing primer and the single-stranded DNA templates in hybridization reaction system, 70-80 DEG C
Lower placement 5-10min, cooled to room temperature complete hybridization；
4-3) it is sequenced：Sequencing reaction system being equipped with, with the step 4-2) obtained hybrid product mix, and it is burnt that counter presses is sequenced
It is carried out according to specific two nucleotide, dGTP feed postitions.
The step 4-2) in, the sequencing primer is one section of one section of sequence with single-stranded DNA templates complete complementary.
In the step 4), each sequencing reaction of two nucleotide obtains the signal strength information of two nucleotide synthesis order-checkings,
The each sequencing reactions of dGTP obtain the signal strength information of single nucleotide acid synthesis order-checking, the sequencing signal strength and synthetic kernel
Thuja acid number is directly proportional, i.e., the sequencing signal strength of each sequencing reaction can be converted into the number of nucleotide synthesis.
In the step 5), the number that the nucleotide of each sequencing reaction acquisitions of dGTP obtained according to burnt sequencing synthesizes is believed
Breath, the information of number are 0≤xi≤1；
The association analysis is：It is assumed that the degree that C is methylated in an easy GC site is xi, wherein 0≤xi≤ 1, that
, in the PCR product Jing Guo bisulf iotate-treated this base sequence, the DNA profiling sequence content containing " C " is xi,
And it is then (1-x to contain " T " the DNA profiling sequence content for not being methylated and being transformed by this basei)；It is sequenced according to dGTP
The information of number of nucleotide synthesis obtained by the reaction, you can the numerical value of xi is obtained.
In the step 2), using traditional bisulfite conversion method or commercially available kit is used, according to it
Operating process is implemented.
In the step 3), mono- primer of PCR used in PCR amplification is 5 ' ends by biotin, amino or acrylamide
Base group modification, with the reaction of the solid phase carrier of surface modification streptavidin, carboxyl or with acrylamide monomer polymerisation system
Standby single-stranded DNA templates.
It is characterized in that：Specific two nucleotide is determined according to DNA profiling analysis segment, including (dCTP+
dTTP)、(dATPαS+dCTP)、(dATPαS+dTTP)。
The beneficial effects of the invention are as follows：
The present invention has the advantages that compared with traditional burnt sequencing：
1) present invention is suitble to the methylation analysis of all PCR product sequences through bisulfite conversion.Compared with tradition
Burnt sequencing analysis method, the present invention can increase substantially the measured length of DNA sequence dna, widen the analysis model of burnt sequencing
It encloses.
2) present invention is suitble to the multi-template PCR product analysis of multiple mixing samples, can directly measure each in mixing sample
The ratio of a DNA profiling can be used for finding from extensive sample, screen nucleic acids marker.
3) present invention directly carries out synthesis order-checking using the natural nucleotide of commercialization, non-marked, it can be at existing
What microarray dataset based on real-time synthesis order-checking carries out.
Description of the drawings
Fig. 1 be the method for the present invention to arbitrarily draft comprising multiple methylation sites PCR product DNA profiling sequence (5 '-
TTTTGGAG (C/T) TTG (C/T) AG (C/T) TTTG (C/T) GTTTTTG (C/T) -3 ', wherein (C/T) is represented through bisulfite
Possibility base information after salt conversion in PCR product), according to specific two nucleotide, dGTP feed postitions carry out burnt sequencing and its
The methylation content association analysis signal in each site；In figure the step of the digital representation sequencing reaction of first, seven rows, second,
What the letter in six row brackets represented the nucleotide of the addition of sequencing reaction writes a Chinese character in simplified form that (" G " represents dGTP, " CA " representative " dCTP+
DATP α S ", the rest may be inferred by analogy), the nucleotide number of the digital representation sequencing reaction synthesis above bracket, the 3rd, the five-element represent
100% methylates (DNA sequence dna 1) and 100% does not methylate (DNA sequence dna 2) sequence；
Fig. 2 adds 5 methylation sites of 2 Gene Promoter CpG Islands of this RAR of proper manners β according to specific two nucleotide, dGTP
Enter mode (being shown in Table 1) progress coke and collection of illustrative plates is sequenced to obtain.
Specific embodiment
A kind of new formed coke sequencing provided by the invention quantitatively detects the method to methylate, is added to simultaneously using two nucleotide
In reaction quickly through " A ", " G ", " T " region in DNA profiling sequence, and the sequencing reaction of single nucleotide acid is applied to measure
" C " content in DNA profiling sequence.Then, " C " content in DNA profiling sequence is measured according to the sequencing reaction of single nucleotide acid, i.e.,
It can determine methylation.
With reference to specific embodiment, the present invention will be further described.
Embodiment 1：
5 methylation sites analysis methods of 2 Gene Promoter CpG Islands of this RAR of proper manners β, specific method include：
(1) by blood sample using traditional protein kinase K and the genome in phenol/chloroform extraction process extraction peripheral blood
DNA；
(2) sodium hydrogensulfite modifier group DNA：According to CpGenome Turbo Bisulfite Modification
The operating process that Kit (Millipore companies) is provided carries out sodium hydrogensulfite processing to genomic DNA, and purifies, recycles modification
DNA afterwards；
(3) PCR amplification：PCR primer:5 '-biotin-GTTGTTTGAGGATTGGGATG-3 ', 5 '-
ATACCCAAACAAACCCTACTC-3 ' and 200mg handles the genomic DNA of processing, 0.2mM dNTP, 1U through sodium hydrogensulfite
Taq archaeal dna polymerases, 1 × amplification buffer, 1.8mM MgCl250 μ L PCR amplification systems carry out 0 and increase, amplification condition is：
95 DEG C of initial denaturation 5min；40 thermal cycles are：94 DEG C of denaturation 30s, 54 DEG C of annealing 45s, 72 DEG C of extension 45s；Last 72 DEG C are prolonged
Stretch 7min；
(4) pcr amplification product and the magnetic bead of Streptavidin modification react, and the DNA chain of modified biological element is made to be fixed to magnetic
It on pearl, is denatured under 0.1M NaOH solutions, loose another DNA chain is removed；Then washing lotion (10mM Tris- are used
Acetate, pH 7.6) washing, the single-stranded DNA templates being fixed；
(5) by sequencing primer 5 '-CCCAAACAAACCCTACT-3 ' and the fixed a copy of it single-stranded DNA templates mould of magnetic bead
Plate is in reaction system ((10mM Tris-HCl, 2M NaCl, 1mM EDTA (sodium ethylene diamine tetracetate), 0.1%Tween 20, pH
7.6) hybridize 5min at 80 DEG C, then cooled to room temperature, complete hybridization；
(6) burnt sequencing reaction system includes 0.1M Tris-Ac (pH 7.7), 2mM EDTA (sodium ethylene diamine tetracetate),
10mM Mg(Ac)2, 0.2%BSA (Bovine serum albumin), 10mM DTT (two mercapto threitols), 10mM APS (phosphosulfates
Gland), 0.4mg/mL PVP (polyvinylpyrrolidone), 4mM D-luciferin (luciferin), 2U/mL ATP
Sulfurylase (adenosine triphosphate sulfurylase), 0.4mM luciferase (luciferase), 2U/mL apyrase VII
(apyrase VII), 2U/mL DNA polymerase is (Klenow fragment, exo -)；Burnt sequencing reaction system
It is mixed for sequencing reaction with above-mentioned (4) hybrid product；
(7) by the reaction system of above-mentioned (6) be placed in burnt sequenator (PSQ 96MA system (Biotage AB,
Uppsala, Sweden)) in, two nucleotide are separately added into successively according to the order of table 1 and carry out burnt sequencing, are obtained by according to successively
The base fragment coding hum pattern 2 of tactic single sequencing reaction is (according to the theoretical calculation of table 1, if single step sequencing is anti-
The number of synthesizing ribonucleotide is answered then to be set for two secondary responses more than 5, complete to avoid synthetic reaction portion)；It is carried according to Fig. 2
Take F1、F3、F5、F7F9Few nucleotide purpose information (peak intensity of 5au is equivalent to a nucleotide) is converted into, x can be obtained1
=0.31, x2=0.42, x3=0.37, x4=0.47, x5=0.51, it is the methylation in five sites.
Table 1.100% methylates or does not methylate the theoretical sequencing result of PCR product DNA profiling
| Sequencing reaction | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| Nucleotide | G | AT | AC | G | CT | G | AC | G | CT | G | AC |
| DNA1 | 1 | 3 | 1 | 1 | 3 | 1 | 1 | 1 | 5 | 1 | 3 |
| DNA2 | 0 | 4 | 3 | 0 | 2 | 0 | 3 | 0 | 5 | 0 | 4 |
Note：AT represents (dATP α S+dTTP), and so on
DNA1 represents 100% methylated DNA fragments：CTTAGCGAGCGCAAGAGCTGTAGGGTTAG
DNA2 represents 100% non-methylated DNA fragments：TTTAGTGAGTGTAAGAGTTGTAGGGTTAG
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (3)
1. it is a kind of it is non-diagnostic for the purpose of burnt sequencing quantitatively detect the method to methylate, it is characterised in that：Step includes：1）DNA is carried
It takes；2）Bisulf iotate-treated；3）PCR amplification；4）Sequencing；5）Association analysis；
The step 4）Sequencing approach be：Burnt sequencing is carried out according to specific two nucleotide, dGTP feed postitions；Wherein, dinuclear
Thuja acid measures serial response and obtains composite number purpose coding information, and dGTP measures serial response and obtains synthesis information of number；
The step 4）Specific method comprise the following steps：
4-1）Prepare single-stranded DNA templates：The magnetic bead of pcr amplification product and Streptavidin package is reacted, biotin modification
DNA chain is fixed on the magnetic bead, is denatured under 0.05-0.2 M NaOH solutions；Loose another DNA chain is removed；
Then with wash liquid, the single-stranded DNA templates being fixed；
4-2）Sequencing primer hybridizes：By sequencing primer and the single-stranded DNA templates in hybridization reaction system, 70-80 °C of decentralization
5-10 min are put, cooled to room temperature completes hybridization；
4-3）Sequencing：Sequencing reaction system is equipped with, with the step 4-2）Obtained hybrid product mixing, coke sequencing is instead according to spy
Fixed two nucleotide, dGTP feed postitions carry out；
The step 4-2）In, the sequencing primer is one section of sequence with single-stranded DNA templates complete complementary；
The step 4）In, each sequencing reaction of two nucleotide obtains the signal strength information of two nucleotide synthesis order-checkings, dGTP
Each sequencing reaction obtains the signal strength information of single nucleotide acid synthesis order-checking, the sequencing signal strength and synthesizing ribonucleotide
Number is directly proportional, i.e., the sequencing signal strength of each sequencing reaction can be converted into the number of nucleotide synthesis；
The step 5）In, the information of number of the nucleotide synthesis of each sequencing reaction acquisitions of dGTP obtained according to burnt sequencing,
The information of number is that bag is 0≤xi≤1；
The association analysis is：It is assumed that the degree that C is methylated in a GC site is xi, wherein 0≤xi≤ 1, then, it is passing through
It crosses in the PCR product of bisulf iotate-treated this base sequence, the DNA profiling sequence content containing " C " is xi, and containing by
It is then (1-x that this base, which is not methylated " T " the DNA profiling sequence content being transformed,i)；It is obtained according to dGTP sequencing reactions
Nucleotide synthesis information of number, you can the numerical value of xi is obtained；
Specific two nucleotide is determined according to DNA profiling analysis segment, including (dCTP+dTTP), (dATP α S+
dCTP)、(dATPαS+dTTP)。
2. it is according to claim 1 it is non-diagnostic for the purpose of burnt sequencing quantitatively detect the method to methylate, it is characterised in that：Institute
State step 2）In, using traditional bisulfite conversion method or use commercially available kit, according to its operating process reality
It applies.
3. it is according to claim 1 it is non-diagnostic for the purpose of burnt sequencing quantitatively detect the method to methylate, it is characterised in that：Institute
State step 3）In, mono- primer of PCR used in PCR amplification is modified for 5 ' ends by biotin, amino or acrylamide group, with
Surface modification streptavidin, the solid phase carrier of carboxyl react or prepare single stranded DNA mould with acrylamide monomer polymerisation
Plate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510621381.XA CN105219851B (en) | 2015-09-25 | 2015-09-25 | A kind of burnt sequencing quantitatively detects the method to methylate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510621381.XA CN105219851B (en) | 2015-09-25 | 2015-09-25 | A kind of burnt sequencing quantitatively detects the method to methylate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105219851A CN105219851A (en) | 2016-01-06 |
| CN105219851B true CN105219851B (en) | 2018-05-25 |
Family
ID=54989117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510621381.XA Expired - Fee Related CN105219851B (en) | 2015-09-25 | 2015-09-25 | A kind of burnt sequencing quantitatively detects the method to methylate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105219851B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624685A (en) * | 2018-01-15 | 2018-10-09 | 新开源弗莱（武汉）生物科技有限公司 | A kind of ABCG2 genetic polymorphism detections kit |
| CN108753958B (en) * | 2018-08-30 | 2021-05-14 | 中国医科大学附属盛京医院 | Detection primer and detection kit based on peripheral blood Sox10 gene methylation and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740824A (en) * | 2014-01-09 | 2014-04-23 | 东南大学 | Method for identifying microbial population by use of real-time synthesis sequencing map of two nucleotides |
| CN104894246A (en) * | 2015-05-21 | 2015-09-09 | 东南大学 | Two-nucleotide synthetic sequencing analysis method for multi-template PCR product |
-
2015
- 2015-09-25 CN CN201510621381.XA patent/CN105219851B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740824A (en) * | 2014-01-09 | 2014-04-23 | 东南大学 | Method for identifying microbial population by use of real-time synthesis sequencing map of two nucleotides |
| CN104894246A (en) * | 2015-05-21 | 2015-09-09 | 东南大学 | Two-nucleotide synthetic sequencing analysis method for multi-template PCR product |
Non-Patent Citations (2)
| Title |
|---|
| DNA甲基化分析方法及相关应用;李金慧等;《癌变.畸变.突变》;20141231;第26卷(第4期);313-316 * |
| 焦测序技术及其在遗传分析中的应用;张晓丹等;《分析化学》;20060430;第34卷(第4期);582-586 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105219851A (en) | 2016-01-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Huang et al. | Profiling DNA methylomes from microarray to genome-scale sequencing | |
| US7217522B2 (en) | Genetic analysis by sequence-specific sorting | |
| Tefferi et al. | Primer on medical genomics part III: microarray experiments and data analysis | |
| US6703228B1 (en) | Methods and products related to genotyping and DNA analysis | |
| CN108884491A (en) | Use of cell-free DNA fragment size to determine copy number variation | |
| CN105238859B (en) | A kind of method for obtaining chicken full-length genome high density SNP marker site | |
| CN108350500A (en) | Nucleic acid for detecting chromosome abnormality and method | |
| KR20020008195A (en) | Microarray-based analysis of polynucleotide sequence variations | |
| CN108504744A (en) | A kind of micro- haplotype genetic marker and its kit for legal medical expert's detection | |
| CN104894246B (en) | A kind of method of two nucleotides synthesis order-checking analysis multi-template PCR primer | |
| CN108715902A (en) | Plum blossom weeping branch character SNP marker and its application | |
| JP2002525127A (en) | Methods and products for genotyping and DNA analysis | |
| CN109706231B (en) | High-throughput SNP (single nucleotide polymorphism) typing method for molecular breeding of litopenaeus vannamei | |
| CN104313168B (en) | A kind of method of Primer selection synthesis sequencing analysis PCR primer haplotype | |
| CN105219851B (en) | A kind of burnt sequencing quantitatively detects the method to methylate | |
| CN115109843A (en) | Functional marking method for detecting variation of multiple rice trait control genes | |
| CN111667883B (en) | Forensic medicine mixed DNA analysis method based on composite micro-haplotype pyrophosphate sequencing atlas analysis | |
| CN117144017B (en) | Molecular marker related to chicken growth traits and application thereof | |
| CN105132573B (en) | A kind of burnt sequencing of two nucleotide synthesis quantitatively detects the method to methylate | |
| CN105256030B (en) | The method that one kind finds new mutation/SNP site based on the burnt sequencing of two nucleotide synthesis | |
| CN114875118B (en) | Methods, kits and devices for determining cell lineage | |
| CN114438233B (en) | Synchronous typing detection system of X chromosome Multi-DIP for genetic relationship identification | |
| CN104762406B (en) | The asynchronous synthesis order-checking analysis PCR primer haplotype approach of two nucleotides of one kind | |
| Myllykangas et al. | Targeted deep resequencing of the human cancer genome using next-generation technologies | |
| CN112522381A (en) | High-throughput method for simultaneously detecting gene mutation and copy number change |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180525 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |