WO2019161810A3 - 基于微孔阵列芯片的单分子文库pcr扩增的基因测序方法 - Google Patents

基于微孔阵列芯片的单分子文库pcr扩增的基因测序方法 Download PDF

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WO2019161810A3
WO2019161810A3 PCT/CN2019/082561 CN2019082561W WO2019161810A3 WO 2019161810 A3 WO2019161810 A3 WO 2019161810A3 CN 2019082561 W CN2019082561 W CN 2019082561W WO 2019161810 A3 WO2019161810 A3 WO 2019161810A3
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pcr amplification
microwell
chip
microwells
dna
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PCT/CN2019/082561
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English (en)
French (fr)
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WO2019161810A2 (zh
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刘亚宝
岳耀斐
张志峰
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张家港万众一芯生物科技有限公司
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Priority to US16/971,967 priority Critical patent/US12059677B2/en
Publication of WO2019161810A2 publication Critical patent/WO2019161810A2/zh
Publication of WO2019161810A3 publication Critical patent/WO2019161810A3/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6869—Methods for sequencing
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    • 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
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0663—Whole sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
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  • Biomedical Technology (AREA)
  • Hematology (AREA)
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  • Dispersion Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

本发明提供一种基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法，包括：步骤一，将PCR扩增液滴加到具有微孔阵列的芯片上，使得芯片上的微孔均是独立的反应空间，有且仅有一个DNA样本片段被固定在微孔内；步骤二，将密封好的微孔芯片于PCR仪上进行PCR扩增；步骤三，扩增结束后，将微孔内的双链DNA变性为单链DNA；步骤四，采用退火的方法，将测序引物S2结合在微孔中的待测DNA样本上；步骤五，测序时，向微孔内按G、C、A、T的顺序加入脱氧核苷酸三磷酸dNTP，配对成功后，DNA样本自身电荷的增加、氢离子或焦磷酸PPI离子的释放引起微孔底部的传感器的信号响应，记录传感器的响应信号并对信号进行处理，得到被测DNA的基因序列信息；步骤六，重复步骤五。
PCT/CN2019/082561 2018-02-22 2019-04-12 基于微孔阵列芯片的单分子文库pcr扩增的基因测序方法 WO2019161810A2 (zh)

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Application Number Priority Date Filing Date Title
US16/971,967 US12059677B2 (en) 2018-02-22 2019-04-12 Method of gene sequencing base on single molecule PCR library preparation on a microwell array chip

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CN201810153345.9A CN110184325A (zh) 2018-02-22 2018-02-22 基于微孔阵列芯片的单分子文库pcr扩增的基因测序方法
CN201810153345.9 2018-02-22

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CN110804653A (zh) * 2019-10-11 2020-02-18 上海小海龟科技有限公司 一种测序方法
CN113140257B (zh) * 2020-01-20 2024-10-22 赛纳生物科技(北京)有限公司 一种基因测序信号去除串扰的方法

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CN101942513A (zh) * 2010-08-27 2011-01-12 深圳博尔美生物科技有限公司 生物芯片及其制备方法
CN102203282A (zh) * 2008-06-25 2011-09-28 生命技术公司 使用大规模fet阵列测量分析物的方法和装置
CN102899244A (zh) * 2012-09-27 2013-01-30 中国科学院半导体研究所 固相-数字pcr芯片及其制作方法

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WO2019161810A2 (zh) 2019-08-29
US12059677B2 (en) 2024-08-13
US20210077995A1 (en) 2021-03-18

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