JP6837073B2 - プライマー伸長中のプライマー−プライマー相互作用の排除 - Google Patents
プライマー伸長中のプライマー−プライマー相互作用の排除 Download PDFInfo
- Publication number
- JP6837073B2 JP6837073B2 JP2018544197A JP2018544197A JP6837073B2 JP 6837073 B2 JP6837073 B2 JP 6837073B2 JP 2018544197 A JP2018544197 A JP 2018544197A JP 2018544197 A JP2018544197 A JP 2018544197A JP 6837073 B2 JP6837073 B2 JP 6837073B2
- Authority
- JP
- Japan
- Prior art keywords
- primer
- nucleic acid
- polymerase
- complementary
- primer extension
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000003993 interaction Effects 0.000 title claims description 13
- 230000008030 elimination Effects 0.000 title description 2
- 238000003379 elimination reaction Methods 0.000 title description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 77
- 108020004707 nucleic acids Proteins 0.000 claims description 76
- 102000039446 nucleic acids Human genes 0.000 claims description 76
- 125000003729 nucleotide group Chemical group 0.000 claims description 53
- 238000000034 method Methods 0.000 claims description 52
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical group O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 44
- 230000000295 complement effect Effects 0.000 claims description 40
- 230000003321 amplification Effects 0.000 claims description 34
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 34
- 229940035893 uracil Drugs 0.000 claims description 22
- 108091034117 Oligonucleotide Proteins 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 16
- 239000011541 reaction mixture Substances 0.000 claims description 12
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 claims description 9
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 claims description 9
- 230000002265 prevention Effects 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 4
- 239000013615 primer Substances 0.000 description 154
- 239000000523 sample Substances 0.000 description 51
- 239000000047 product Substances 0.000 description 22
- 239000000539 dimer Substances 0.000 description 20
- 108020004414 DNA Proteins 0.000 description 12
- 230000010076 replication Effects 0.000 description 12
- 238000012163 sequencing technique Methods 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- 108060002716 Exonuclease Proteins 0.000 description 9
- 102000013165 exonuclease Human genes 0.000 description 9
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 7
- 108091093078 Pyrimidine dimer Proteins 0.000 description 7
- 239000013635 pyrimidine dimer Substances 0.000 description 7
- 238000000137 annealing Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000002194 synthesizing effect Effects 0.000 description 6
- 102000040350 B family Human genes 0.000 description 5
- 108091072128 B family Proteins 0.000 description 5
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 5
- 241000589596 Thermus Species 0.000 description 5
- 241000589499 Thermus thermophilus Species 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000003155 DNA primer Substances 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000037452 priming Effects 0.000 description 4
- 241000203069 Archaea Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000001668 nucleic acid synthesis Methods 0.000 description 3
- 239000002987 primer (paints) Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108010002747 Pfu DNA polymerase Proteins 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
- ASJWEHCPLGMOJE-LJMGSBPFSA-N ac1l3rvh Chemical class N1C(=O)NC(=O)[C@@]2(C)[C@@]3(C)C(=O)NC(=O)N[C@H]3[C@H]21 ASJWEHCPLGMOJE-LJMGSBPFSA-N 0.000 description 2
- 125000002015 acyclic group Chemical group 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 108010055863 gene b exonuclease Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 108091093088 Amplicon Proteins 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
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 102000016559 DNA Primase Human genes 0.000 description 1
- 108010092681 DNA Primase Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102100030569 Nuclear receptor corepressor 2 Human genes 0.000 description 1
- 101710153660 Nuclear receptor corepressor 2 Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000001218 Rec A Recombinases Human genes 0.000 description 1
- 108010055016 Rec A Recombinases Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000204652 Thermotoga Species 0.000 description 1
- 241000589500 Thermus aquaticus Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 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
- 238000001574 biopsy Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000010485 coping Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- UPUOLJWYFICKJI-UHFFFAOYSA-N cyclobutane;pyrimidine Chemical class C1CCC1.C1=CN=CN=C1 UPUOLJWYFICKJI-UHFFFAOYSA-N 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000003811 finger Anatomy 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 238000001782 photodegradation Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010032203 pyrimidine(6-4)pyrimidone photolyase Proteins 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000003813 thumb Anatomy 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Images
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
-
- 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
- C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
- C12Q1/6855—Ligating adaptors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/101—Modifications characterised by incorporating non-naturally occurring nucleotides, e.g. inosine
-
- 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
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/186—Modifications characterised by incorporating a non-extendable or blocking moiety
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
用語「ポリメラーゼ」は、天然(その天然種から精製される)であってもまたは組換え体(形質転換宿主で産生され、そしてそこから精製される)であってもよい核酸ポリメラーゼを指す。本発明の背景において、ポリメラーゼは、元来のアミノ酸配列、または本発明の方法にしたがって修飾塩基で複製を失速させる能力をポリメラーゼが維持する限り、修飾アミノ酸配列を有することも可能である。
分子反転プローブ(MIP)は、次世代配列決定適用において、ターゲット配列を濃縮させるために用いられてきている。用いるプローブプールは、数百そしてさらに数千のターゲットアンプリコンを生成しうる。しかし、数百または数千の他のプローブの背景においてよく働くプローブを設計することは容易ではない。これは、最終ライブラリーにおいて、望ましくない産物を生成する、プローブ間の相互作用が起こりうるためである。この状況は、これらのプローブにおいてユニークな識別子配列を用いた際に悪化する可能性もあり、これはこれらが配列決定を通じた分子同定を可能にするために、プローブ内に操作されるランダム配列であるためである。分子反転プローブ内へのウラシル塩基の取り込みは、2つのプローブが相互作用した際に、ポリメラーゼ伸長をブロッキングすることにより、望ましくない産物の生成を減少させるはずである。
Claims (8)
- プライマー−プライマー相互作用を減少させつつ、試料中のターゲット核酸を増幅する方法であって:
a.試料を、核酸ポリメラーゼ、およびポリメラーゼによるヌクレオチド取り込みを失速させる少なくとも1つの修飾ヌクレオチドを含む、ターゲット核酸に相補的な第一のプライマーと接触させて、プライマー伸長産物を生成する、プライマー伸長工程;
b.該プライマー伸長産物を二本鎖アダプターに連結する;および
c.該試料を、プライマー伸長産物に相補的な第二のプライマーと接触させる、指数関数的増幅工程
を含み、
該プライマーが、ターゲット配列に非相補的なリンカー配列によって分離されている、ターゲット配列に相補的な2つのアームからなる一本鎖オリゴヌクレオチドである、
前記方法。 - 前記アダプターおよび第一のプライマーが、普遍的増幅プライマーの結合部位を含み、そして前記第二のプライマーが普遍的プライマーである、請求項1の方法。
- 前記指数関数的増幅工程が、修飾ヌクレオチドに耐性であるポリメラーゼを利用する、請求項1または2の方法。
- 工程cの前に精製工程をさらに含み、ここで第一のプライマーおよびテンプレート核酸が、プライマー伸長産物から分離される、請求項1〜3のいずれか1項の方法。
- 前記修飾ヌクレオチドがウラシルである、請求項1〜4のいずれか1項の方法。
- 工程cの前にキャリーオーバー防止工程をさらに含み、ここで試料をウラシルDNAグリコシラーゼと接触させる、請求項5の方法。
- 請求項1〜6のいずれか1項の方法にしたがって、試料におけるターゲット核酸を増幅するためのキットであって、
核酸ポリメラーゼ、
ポリメラーゼによるヌクレオチド取り込みを失速させる少なくとも1つの修飾ヌクレオチドを含む、プライマー伸長産物を生成するための、ターゲット核酸に相補的な第一のプライマー、
該プライマー伸長産物に連結するための二本鎖アダプター、および
該プライマー伸長産物に相補的な第二のプライマー、
を含み、
該プライマーが、ターゲット配列に非相補的なリンカー配列によって分離されている、ターゲット配列に相補的な2つのアームからなる一本鎖オリゴヌクレオチドである、
前記キット。 - 請求項1〜6のいずれか1項の方法にしたがって、試料におけるターゲット核酸を増幅するための反応混合物であって、
核酸ポリメラーゼ、および
ポリメラーゼによるヌクレオチド取り込みを失速させる少なくとも1つの修飾ヌクレオチドを含む、プライマー伸長産物を生成するための、ターゲット核酸に相補的な第一のプライマー、
を含み、
該プライマーが、ターゲット配列に非相補的なリンカー配列によって分離されている、ターゲット配列に相補的な2つのアームからなる一本鎖オリゴヌクレオチドである、
前記反応混合物。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662299988P | 2016-02-25 | 2016-02-25 | |
US62/299,988 | 2016-02-25 | ||
PCT/EP2017/053922 WO2017144457A1 (en) | 2016-02-25 | 2017-02-21 | Elimination of primer-primer interactions during primer extension |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2019506172A JP2019506172A (ja) | 2019-03-07 |
JP6837073B2 true JP6837073B2 (ja) | 2021-03-03 |
Family
ID=58098620
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2018544197A Active JP6837073B2 (ja) | 2016-02-25 | 2017-02-21 | プライマー伸長中のプライマー−プライマー相互作用の排除 |
Country Status (6)
Country | Link |
---|---|
US (2) | US10961567B2 (ja) |
EP (1) | EP3420100B1 (ja) |
JP (1) | JP6837073B2 (ja) |
CN (1) | CN108699595B (ja) |
ES (1) | ES2874275T3 (ja) |
WO (1) | WO2017144457A1 (ja) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6681804B2 (ja) * | 2016-03-30 | 2020-04-15 | 大研医器株式会社 | 変異プライマーの設計方法 |
WO2019121842A1 (en) * | 2017-12-21 | 2019-06-27 | F. Hoffmann-La Roche Ag | Target enrichment by unidirectional dual probe primer extension |
CN113862339B (zh) * | 2021-12-01 | 2022-03-22 | 广州滴纳生物科技有限公司 | 核酸组合产品、检测试剂盒和扩增靶核酸的方法 |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5035996A (en) * | 1989-06-01 | 1991-07-30 | Life Technologies, Inc. | Process for controlling contamination of nucleic acid amplification reactions |
US5683896A (en) | 1989-06-01 | 1997-11-04 | Life Technologies, Inc. | Process for controlling contamination of nucleic acid amplification reactions |
CA2426824A1 (en) * | 2000-10-24 | 2002-07-25 | The Board Of Trustees Of The Leland Stanford Junior University | Direct multiplex characterization of genomic dna |
US8507662B2 (en) | 2001-01-19 | 2013-08-13 | General Electric Company | Methods and kits for reducing non-specific nucleic acid amplification |
US7629152B2 (en) * | 2002-03-01 | 2009-12-08 | Integrated Dna Technologies, Inc. | Methods for amplifying polymeric nucleic acids |
US7211382B2 (en) * | 2002-04-09 | 2007-05-01 | Orchid Cellmark Inc. | Primer extension using modified nucleotides |
US7517978B1 (en) * | 2004-04-14 | 2009-04-14 | Applied Biosystems, Llc | Modified oligonucleotides and applications thereof |
CN101842494B (zh) * | 2007-07-03 | 2013-05-29 | 吉纳珀莱有限公司 | 用于改进的核酸扩增反应的嵌合引物 |
AU2011296209B2 (en) * | 2010-08-30 | 2015-07-02 | Dow Agrosciences Llc | Sugarcane bacilliform viral (SCBV) enhancer and its use in plant functional genomics |
JP2015522277A (ja) * | 2012-06-28 | 2015-08-06 | カルドラ ヘルス リミテッドCaldera Health Ltd | 前立腺癌の診断方法と診断用物質 |
CN104685064A (zh) | 2012-07-24 | 2015-06-03 | 纳特拉公司 | 高度复合pcr方法和组合物 |
CN103114131B (zh) * | 2012-11-30 | 2018-10-02 | 珠海市坤元农业科技有限公司 | 一种引物中部序列干扰pcr技术 |
WO2014110272A1 (en) | 2013-01-09 | 2014-07-17 | The Penn State Research Foundation | Low sequence bias single-stranded dna ligation |
-
2017
- 2017-02-21 ES ES17706465T patent/ES2874275T3/es active Active
- 2017-02-21 US US16/080,151 patent/US10961567B2/en active Active
- 2017-02-21 JP JP2018544197A patent/JP6837073B2/ja active Active
- 2017-02-21 WO PCT/EP2017/053922 patent/WO2017144457A1/en active Application Filing
- 2017-02-21 EP EP17706465.6A patent/EP3420100B1/en active Active
- 2017-02-21 CN CN201780012552.6A patent/CN108699595B/zh active Active
-
2021
- 2021-02-23 US US17/183,255 patent/US11946099B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
CN108699595B (zh) | 2022-11-04 |
US20210254146A1 (en) | 2021-08-19 |
US10961567B2 (en) | 2021-03-30 |
EP3420100B1 (en) | 2021-04-07 |
US20190048410A1 (en) | 2019-02-14 |
WO2017144457A1 (en) | 2017-08-31 |
ES2874275T3 (es) | 2021-11-04 |
JP2019506172A (ja) | 2019-03-07 |
CN108699595A (zh) | 2018-10-23 |
US11946099B2 (en) | 2024-04-02 |
EP3420100A1 (en) | 2019-01-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11274340B2 (en) | Methods, compositions and kits for small RNA capture, detection and quantification | |
CN110191961B (zh) | 制备经不对称标签化的测序文库的方法 | |
JP6525473B2 (ja) | 複製物配列決定リードを同定するための組成物および方法 | |
JP6847496B2 (ja) | 単一プローブプライマー伸長による標的濃縮 | |
US20060211000A1 (en) | Methods, compositions, and kits for detection of microRNA | |
EP2272976A1 (en) | Method for differentiation of polynucleotide strands | |
EP2365079A1 (en) | Processes using dual specificity oligonucleotide and dual specificity oligonucleotide | |
EP3601593B1 (en) | Universal hairpin primers | |
US11946099B2 (en) | Elimination of primer-primer interactions during primer extension | |
KR102398479B1 (ko) | 카피수 보존 rna 분석 방법 | |
CN111801427B (zh) | 用于单分子的单链环状dna模板的产生 | |
JP7134186B2 (ja) | Rnaおよびdnaからの核酸ライブラリーの作製 | |
AU2006226873B2 (en) | Nucleic acid detection | |
CN113557298A (zh) | 核酸的生成和扩增 | |
KR20230124636A (ko) | 멀티플렉스 반응에서 표적 서열의 고 감응성 검출을위한 조성물 및 방법 | |
CN116113709A (zh) | 基因分型和核酸测序中的伪互补碱基 | |
JP6983906B2 (ja) | ライブラリーの定量および定性 | |
US20240209414A1 (en) | Novel nucleic acid template structure for sequencing | |
WO2024112758A1 (en) | High-throughput amplification of targeted nucleic acid sequences |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20180921 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20190723 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20190729 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20191028 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20191108 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200417 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20200716 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200916 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20210204 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210208 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6837073 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |