CN105648537A - Dna5-甲基胞嘧啶与5-羟甲基胞嘧啶基因图谱测序方法 - Google Patents
Dna5-甲基胞嘧啶与5-羟甲基胞嘧啶基因图谱测序方法 Download PDFInfo
- Publication number
- CN105648537A CN105648537A CN201610119096.2A CN201610119096A CN105648537A CN 105648537 A CN105648537 A CN 105648537A CN 201610119096 A CN201610119096 A CN 201610119096A CN 105648537 A CN105648537 A CN 105648537A
- Authority
- CN
- China
- Prior art keywords
- dna
- hydroxymethyl cytosine
- methylcystein
- dna5
- cytosine
- 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.)
- Granted
Links
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 title claims abstract description 116
- 238000000034 method Methods 0.000 title claims abstract description 24
- 108091030081 DNA N(4)-methylcytosine Proteins 0.000 title abstract 2
- 238000012268 genome sequencing Methods 0.000 title abstract 2
- 238000006062 fragmentation reaction Methods 0.000 claims abstract description 46
- 238000013467 fragmentation Methods 0.000 claims abstract description 41
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims abstract description 40
- 238000002372 labelling Methods 0.000 claims abstract description 31
- 239000007790 solid phase Substances 0.000 claims abstract description 29
- 238000012163 sequencing technique Methods 0.000 claims abstract description 21
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000012634 fragment Substances 0.000 claims abstract description 15
- 238000009826 distribution Methods 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 28
- 238000005259 measurement Methods 0.000 claims description 25
- 238000013507 mapping Methods 0.000 claims description 24
- 238000000746 purification Methods 0.000 claims description 23
- 239000011324 bead Substances 0.000 claims description 22
- 229940104302 cytosine Drugs 0.000 claims description 19
- 230000005291 magnetic effect Effects 0.000 claims description 19
- 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 18
- 229960002685 biotin Drugs 0.000 claims description 13
- 239000011616 biotin Substances 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 10
- 235000020958 biotin Nutrition 0.000 claims description 9
- 210000001519 tissue Anatomy 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 8
- 210000001124 body fluid Anatomy 0.000 claims description 8
- 239000010839 body fluid Substances 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 claims description 8
- DVOOXRTYGGLORL-VKHMYHEASA-N (2r)-2-(methylamino)-3-sulfanylpropanoic acid Chemical compound CN[C@@H](CS)C(O)=O DVOOXRTYGGLORL-VKHMYHEASA-N 0.000 claims description 7
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 7
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical group [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 claims description 7
- 238000005498 polishing Methods 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 108010091086 Recombinases Proteins 0.000 claims description 6
- 102000018120 Recombinases Human genes 0.000 claims description 6
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 6
- 239000012148 binding buffer Substances 0.000 claims description 6
- 102000004316 Oxidoreductases Human genes 0.000 claims description 5
- 108090000854 Oxidoreductases Proteins 0.000 claims description 5
- 210000004369 blood Anatomy 0.000 claims description 5
- 239000008280 blood Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 229920000936 Agarose Polymers 0.000 claims description 3
- 206010003445 Ascites Diseases 0.000 claims description 3
- 206010048612 Hydrothorax Diseases 0.000 claims description 3
- 241001597008 Nomeidae Species 0.000 claims description 3
- 206010036790 Productive cough Diseases 0.000 claims description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 3
- 210000000941 bile Anatomy 0.000 claims description 3
- 150000001720 carbohydrates Chemical class 0.000 claims description 3
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 3
- 102000038379 digestive enzymes Human genes 0.000 claims description 3
- 108091007734 digestive enzymes Proteins 0.000 claims description 3
- 210000003608 fece Anatomy 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 229920002521 macromolecule Polymers 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 210000003463 organelle Anatomy 0.000 claims description 3
- 210000000496 pancreas Anatomy 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- 210000003802 sputum Anatomy 0.000 claims description 3
- 208000024794 sputum Diseases 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 3
- 125000004122 cyclic group Chemical group 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- HWPZZUQOWRWFDB-UHFFFAOYSA-N 1-methylcytosine Chemical compound CN1C=CC(N)=NC1=O HWPZZUQOWRWFDB-UHFFFAOYSA-N 0.000 claims 6
- 238000003752 polymerase chain reaction Methods 0.000 abstract 3
- 238000007481 next generation sequencing Methods 0.000 abstract 2
- 238000006243 chemical reaction Methods 0.000 description 35
- 239000000376 reactant Substances 0.000 description 30
- 239000000203 mixture Substances 0.000 description 14
- 229920001213 Polysorbate 20 Polymers 0.000 description 13
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 13
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 239000000872 buffer Substances 0.000 description 9
- 238000004140 cleaning Methods 0.000 description 9
- 238000005859 coupling reaction Methods 0.000 description 8
- 238000010438 heat treatment Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 239000011541 reaction mixture Substances 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 239000004094 surface-active agent Substances 0.000 description 6
- 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 5
- 150000001875 compounds Chemical class 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 102000012410 DNA Ligases Human genes 0.000 description 4
- 108010061982 DNA Ligases Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 101710163270 Nuclease Proteins 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- -1 alpha-glucosyl Chemical group 0.000 description 4
- 125000003368 amide group Chemical group 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000008836 DNA modification Effects 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 150000001993 dienes Chemical class 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 230000005298 paramagnetic effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- MJEQLGCFPLHMNV-UHFFFAOYSA-N 4-amino-1-(hydroxymethyl)pyrimidin-2-one Chemical compound NC=1C=CN(CO)C(=O)N=1 MJEQLGCFPLHMNV-UHFFFAOYSA-N 0.000 description 1
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- YLSUUMDTTUVTGP-UHFFFAOYSA-N [6-(azidomethyl)-3,4,5-trihydroxyoxan-2-yl] [[5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] hydrogen phosphate Chemical group O1C(N2C(NC(=O)C=C2)=O)C(O)C(O)C1COP(O)(=O)OP(O)(=O)OC1OC(CN=[N+]=[N-])C(O)C(O)C1O YLSUUMDTTUVTGP-UHFFFAOYSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- VOAXAOULFRTTAM-UHFFFAOYSA-N chloroform phenol Chemical compound C1(=CC=CC=C1)O.C(Cl)(Cl)Cl.C1(=CC=CC=C1)O.C1(=CC=CC=C1)O VOAXAOULFRTTAM-UHFFFAOYSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000019975 dosage compensation by inactivation of X chromosome Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Bioinformatics & Computational Biology (AREA)
Abstract
Description
反应物 | 体积(uL) |
Nuclease free water | 5 |
Ligation Buffer | 30 |
DNA Ligase | 10 |
反应物 | 体积(uL) |
连接反应混合物 | 35 |
接头引物 | 5 |
反应物 | 终浓度 |
UDP-Glu | 50uM |
βGT | 1uM |
Mg2+ | 25mM |
HEPESpH=8.0 | 50mM |
DNA(20uL) |
反应物 | 终浓度 |
UDP–N3-Glu | 50uM |
βGT | 1uM |
mTet1 | 3uM6 --> |
Mg2+ | 10mM |
HEPES pH=8.0 | 50mM |
Fe2+ | 75uM |
Ascorbic Acid | 2mM |
Dithiothreitol | 1mM |
DNA(20uL) |
反应物 | 终浓度 |
DBCO-Biotin | 2mM |
DNA(20uL) |
反应物 | 体积(uL) |
2XPCR master mix | 25 |
PCR primer | 1.25 |
反应物 | 体积(uL) |
Nuclease free water | 5 |
Ligation Buffer | 30 |
DNA Ligase | 10 |
反应物 | 体积(uL) |
连接反应混合物 | 35 |
接头引物 | 5 |
反应物 | 终浓度 |
UDP–N3-Glu | 50uM |
βGT | 1uM |
Mg2+ | 25mM |
HEPES pH=8.0 | 50mM |
DNA(20uL) |
反应物 | 终浓度 |
DBCO-Biotin | 2mM |
DNA(20uL) |
反应物 | 体积(uL) |
2XPCR master mix | 25 |
PCR primer | 1.25 |
反应物 | 体积(uL) |
Nuclease free water | 5 |
Ligation Buffer | 30 |
DNA Ligase | 10 |
反应物 | 体积(uL) |
连接反应混合物 | 35 |
接头引物 | 5 |
反应物 | 终浓度 |
UDP-Glu | 50uM |
βGT | 1uM |
Mg2+ | 25mM |
HEPES pH=8.0 | 50mM |
DNA(20uL) |
反应物 | 终浓度11 --> |
UDP–N3-Glu | 50uM |
βGT | 1uM |
mTet1 | 3uM |
Mg2+ | 10mM |
HEPES pH=8.0 | 50mM |
Fe2+ | 75uM |
Ascorbic Acid | 2mM |
Dithiothreitol | 1mM |
DNA(20uL) |
反应物 | 终浓度 |
DBCO-Biotin | 2mM |
DNA(20uL) |
反应物 | 体积(uL) |
2XPCR master mix | 25 |
PCR primer | 1.25 |
反应物 | 体积(uL) |
Nuclease free water | 5 |
Ligation Buffer | 30 |
DNA Ligase | 10 |
反应物 | 体积(uL) |
连接反应混合物 | 35 |
接头引物 | 5 |
反应物 | 终浓度 |
UDP–N3-Glu | 50uM |
βGT | 1uM |
Mg2+ | 25mM |
HEPES pH=8.0 | 50mM |
DNA(20uL) |
反应物 | 终浓度 |
DBCO-Biotin | 2mM |
DNA(20uL) |
反应物 | 体积(uL) |
2XPCR master mix | 25 |
PCR primer | 1.25 |
Claims (10)
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610119096.2A CN105648537B (zh) | 2016-03-02 | 2016-03-02 | Dna5-甲基胞嘧啶与5-羟甲基胞嘧啶基因图谱测序方法 |
PCT/CN2016/075863 WO2017147945A1 (zh) | 2016-03-02 | 2016-03-08 | Dna5-甲基胞嘧啶与5-羟甲基胞嘧啶基因图谱测序方法 |
TW106102252A TWI626315B (zh) | 2016-03-02 | 2017-01-20 | DNA 5-methylcytosine and 5-hydroxymethylcytosine gene mapping method |
US15/497,247 US11162139B2 (en) | 2016-03-02 | 2017-04-26 | Method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610119096.2A CN105648537B (zh) | 2016-03-02 | 2016-03-02 | Dna5-甲基胞嘧啶与5-羟甲基胞嘧啶基因图谱测序方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105648537A true CN105648537A (zh) | 2016-06-08 |
CN105648537B CN105648537B (zh) | 2018-06-29 |
Family
ID=56492951
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610119096.2A Active CN105648537B (zh) | 2016-03-02 | 2016-03-02 | Dna5-甲基胞嘧啶与5-羟甲基胞嘧啶基因图谱测序方法 |
Country Status (3)
Country | Link |
---|---|
CN (1) | CN105648537B (zh) |
TW (1) | TWI626315B (zh) |
WO (1) | WO2017147945A1 (zh) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107164508A (zh) * | 2017-06-16 | 2017-09-15 | 上海易毕恩基因科技有限公司 | 用于检测肝癌的基因标志物及其用途 |
US9816986B2 (en) | 2008-09-26 | 2017-11-14 | Children's Medical Center Corporation | Detection of 5-hydroxymethylcytosine by glycosylation |
US9822394B2 (en) | 2014-02-24 | 2017-11-21 | Cambridge Epigenetix Limited | Nucleic acid sample preparation |
CN107385051A (zh) * | 2017-08-04 | 2017-11-24 | 上海易毕恩生物技术有限公司 | 用于检测肝肿瘤良恶性的基因标志物、试剂盒及检测方法 |
WO2018036537A1 (zh) * | 2016-08-24 | 2018-03-01 | 中国科学院上海生命科学研究院 | Cmd1酶催化的5-甲基胞嘧啶核酸的新修饰及其应用 |
US10428381B2 (en) | 2011-07-29 | 2019-10-01 | Cambridge Epigenetix Limited | Methods for detection of nucleotide modification |
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