CN111500676A - 一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法 - Google Patents
一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法 Download PDFInfo
- Publication number
- CN111500676A CN111500676A CN202010316713.4A CN202010316713A CN111500676A CN 111500676 A CN111500676 A CN 111500676A CN 202010316713 A CN202010316713 A CN 202010316713A CN 111500676 A CN111500676 A CN 111500676A
- Authority
- CN
- China
- Prior art keywords
- rna
- oligonucleotide
- primer
- upstream
- downstream
- 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
- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 63
- 238000001514 detection method Methods 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 36
- 239000002299 complementary DNA Substances 0.000 title claims abstract description 28
- 238000010839 reverse transcription Methods 0.000 title claims description 16
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 36
- 108020004414 DNA Proteins 0.000 claims abstract description 26
- 102000053602 DNA Human genes 0.000 claims abstract description 16
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 15
- 230000000295 complement effect Effects 0.000 claims abstract description 13
- 239000012634 fragment Substances 0.000 claims abstract description 13
- 150000004712 monophosphates Chemical group 0.000 claims abstract description 5
- 102000012410 DNA Ligases Human genes 0.000 claims abstract description 4
- 108010061982 DNA Ligases Proteins 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 22
- 238000011529 RT qPCR Methods 0.000 claims description 12
- 238000012163 sequencing technique Methods 0.000 claims description 9
- 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 claims description 7
- 238000012165 high-throughput sequencing Methods 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 5
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 5
- 102000003960 Ligases Human genes 0.000 claims description 5
- 108090000364 Ligases Proteins 0.000 claims description 5
- 238000001727 in vivo Methods 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 abstract description 12
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 44
- 239000000243 solution Substances 0.000 description 9
- 238000002156 mixing Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 3
- 238000006209 dephosphorylation reaction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 description 2
- 102100024319 Intestinal-type alkaline phosphatase Human genes 0.000 description 2
- 101710184243 Intestinal-type alkaline phosphatase Proteins 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000030609 dephosphorylation Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 101150022630 prp5 gene Proteins 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 1
- 102100023123 Mucin-16 Human genes 0.000 description 1
- 230000007022 RNA scission Effects 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 101100191561 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PRP3 gene Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing 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
- C12Q1/6806—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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims (8)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010316713.4A CN111500676B (zh) | 2020-04-21 | 2020-04-21 | 一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010316713.4A CN111500676B (zh) | 2020-04-21 | 2020-04-21 | 一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111500676A true CN111500676A (zh) | 2020-08-07 |
CN111500676B CN111500676B (zh) | 2022-09-09 |
Family
ID=71872772
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010316713.4A Active CN111500676B (zh) | 2020-04-21 | 2020-04-21 | 一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111500676B (zh) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001085976A1 (en) * | 2000-05-08 | 2001-11-15 | The Regents Of The University Of California | Methods for making nucleic acids |
WO2009024599A1 (en) * | 2007-08-23 | 2009-02-26 | Novartis Ag | Methods for detecting oligonucleotides |
CN103074419A (zh) * | 2012-09-27 | 2013-05-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | 基于生物芯片的检测核酸降解组mRNA的通用探针方法 |
CN104428415A (zh) * | 2012-07-10 | 2015-03-18 | 莱克斯奥根有限公司 | 5’保护依赖性扩增 |
CN107429296A (zh) * | 2015-03-13 | 2017-12-01 | 生命技术公司 | 捕获、检测和定量小rna的方法、组合物与试剂盒 |
WO2018016885A1 (ko) * | 2016-07-21 | 2018-01-25 | 주식회사 하임바이오텍 | 알앤에이 검출용 키트 및 방법 |
CN108384831A (zh) * | 2018-02-13 | 2018-08-10 | 塔里木大学 | 具有4至10个核苷酸单体的寡聚核苷酸的检测方法 |
-
2020
- 2020-04-21 CN CN202010316713.4A patent/CN111500676B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001085976A1 (en) * | 2000-05-08 | 2001-11-15 | The Regents Of The University Of California | Methods for making nucleic acids |
WO2009024599A1 (en) * | 2007-08-23 | 2009-02-26 | Novartis Ag | Methods for detecting oligonucleotides |
CN104428415A (zh) * | 2012-07-10 | 2015-03-18 | 莱克斯奥根有限公司 | 5’保护依赖性扩增 |
CN103074419A (zh) * | 2012-09-27 | 2013-05-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | 基于生物芯片的检测核酸降解组mRNA的通用探针方法 |
CN107429296A (zh) * | 2015-03-13 | 2017-12-01 | 生命技术公司 | 捕获、检测和定量小rna的方法、组合物与试剂盒 |
WO2018016885A1 (ko) * | 2016-07-21 | 2018-01-25 | 주식회사 하임바이오텍 | 알앤에이 검출용 키트 및 방법 |
CN108384831A (zh) * | 2018-02-13 | 2018-08-10 | 塔里木大学 | 具有4至10个核苷酸单体的寡聚核苷酸的检测方法 |
Also Published As
Publication number | Publication date |
---|---|
CN111500676B (zh) | 2022-09-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1644520B1 (en) | Method for selective detection of a target nucleic acid | |
US9856521B2 (en) | Ligation assays in liquid phase | |
US8314220B2 (en) | Methods compositions, and kits for detection of microRNA | |
EP1747285B1 (en) | Method for amplifying specific nucleic acids in parallel | |
JP6739339B2 (ja) | カバーされた配列変換dnaおよび検出方法 | |
CA2567735C (en) | Rapid production of double-stranded target dna molecules | |
EP1047794A2 (en) | Method for the detection or nucleic acid of nucleic acid sequences | |
CN102643910B (zh) | 非对称多色荧光发夹探针链反应在病原菌检测中的应用 | |
US20060003337A1 (en) | Detection of small RNAS | |
WO2015196120A1 (en) | Methods and compositions for detecting polynucleotides and fragments thereof | |
KR20180053743A (ko) | 짧은 호모폴리머릭 반복서열의 개선된 검출법 | |
WO2014134320A1 (en) | Methods, compositions and systems for the analysis of nucleic acid molecules | |
EP3378948B1 (en) | Method for quantifying target nucleic acid and kit therefor | |
EP2971124B1 (en) | Methods for amplification of nucleic acids utilizing clamp oligonuleotides | |
WO2019084245A1 (en) | METHODS AND COMPOSITIONS FOR PREPARING NUCLEIC ACID LIBRARIES | |
CN113913499A (zh) | 利用Cas12j效应蛋白检测目标突变的方法 | |
CN111500676B (zh) | 一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法 | |
CN109852670A (zh) | 一种高特异性核酸检测试剂及其使用方法 | |
CN102559856A (zh) | 去除测序文库中的载体片段的方法 | |
CN113817723B (zh) | 一种多核苷酸及其标准品、试剂盒与用途 | |
WO2018081666A1 (en) | Methods of single dna/rna molecule counting | |
CN116179652A (zh) | 基于LAMP联合Cas13a核酸酶的检测痕量核酸的方法以及应用 | |
JP7333171B2 (ja) | Rna検出方法、rna検出用核酸及びrna検出用キット | |
CN114606298A (zh) | 一种检测样品中一种或多种核酸分子扩增产物长度的方法 | |
Lu et al. | High-throughput approaches for miRNA expression analysis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
CB03 | Change of inventor or designer information | ||
CB03 | Change of inventor or designer information |
Inventor after: Qin Shaowei Inventor after: Zhao Lifeng Inventor after: Li Cailin Inventor before: Zhao Lifeng Inventor before: Qin Shaowei Inventor before: Li Cailin |
|
GR01 | Patent grant | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20231116 Address after: No. 358, 26th Floor, Building 1, Yifeng Xinduhui, No.16 Production Road, Huiji District, Jinshui District, Zhengzhou City, Henan Province, 450053 Patentee after: Henan Baiaotai Biotechnology Co.,Ltd. Address before: 541006 No.26 Liangfeng Road, Yanshan District, Guilin City, Guangxi Zhuang Autonomous Region Patentee before: GUILIN TOURISM University |