KR960704066A - 핵산 증폭을 강화하는 방법(methods for enhancing nucleic acid amplification) - Google Patents

핵산 증폭을 강화하는 방법(methods for enhancing nucleic acid amplification)

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Publication number
KR960704066A
KR960704066A KR1019960700342A KR19960700342A KR960704066A KR 960704066 A KR960704066 A KR 960704066A KR 1019960700342 A KR1019960700342 A KR 1019960700342A KR 19960700342 A KR19960700342 A KR 19960700342A KR 960704066 A KR960704066 A KR 960704066A
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primer
nucleic acid
primers
acid strand
dna polymerase
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KR1019960700342A
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English (en)
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KR100189229B1 (ko
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비. 라이더 토마스
알. 빌야드 엘리자베스
다타굽타 나니브휴샨
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다니엘 엘. 카시안
젠-프로브 인코포레이티드
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    • 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
    • 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
    • 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
    • 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
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Abstract

본 발명은 테스트 샘플내의 핵산을 증폭하는 방법으로, 이는 테스트 샘플로부터의 핵산 가닥 및 최소한 3개의 올리고누클레오티드 프라이머를 동시에 접촉시키는 단계를 포함한다. 최소한 하나의 프라이머는 프로모터-프라이머이고, 최소한 하나의 다른 프라이머는 핵산 가닥에 상보적인 프라이머이며, 다른 하나의 프라이머는 핵산 가닥에 상보적인 가닥에 상보적인 프라이머이다. 본 발명에 따른 방법은 핵산 가닥 및 RNA-유도성 및/또는 DNA-유도성 DNA 중합효소 활성, RNA 중합효소 활성 및 RNAse H활성을 가지는 하나 이상의 단백질을 프라이머-신장 조건하에서 접촉시켜 필수적으로 상온에서 핵산 가닥의 표적 영역을 증폭하는 단계를 더 포함한다.

Description

핵산 증폭을 강화하는 방법(METHODS FOR ENHANCING NUCLEIC ACID AMPLIFICATION)
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 폴1 표적 영역의 구조에 관한 프라이머의 위치를 도시한 도면.
제2도는 증폭 개시 방법. IMI. IM2 및 IM3 프로토콜을 도시한 도면.
제3도는 외부 T7 프라이머의 신장에 의한 개시를 위한 가능한 설계를 도시한 도면.
제4도는 표적-특이적인 개시를 보조하여 더 효율적으로 만드는 4009 프라이머의 후속 신장에 의한 T74116 프라이머 신장 생성물의 잠재적인 가닥 배치 활성을 도시한 도면이다.

Claims (20)

  1. 테스트 샘플에서의 핵산 가닥, 최소한 3개의 올리고누클레오티드 프라이머, 프로모터-프라이머로 최소한 1개의 프라이머, 및 핵산 가닥과 상보적인 가닥에 상보적인 최소한 1개의 다른 프랴이머를 동시에 접촉시키는 제1접촉단계; 및 상기 핵산 가닥 및 프라이머를 RNA-유도성 및/또는 DNA-유도성 DNA 중합효소 활성, RNA 중합효소 활성 및 RNAse H 활성을 가지는 하나 이상의 단백질과 프라이머-신장 조건에서 접촉시켜 상온에서 상기 핵산을 증폭시키는 제2접촉단계를 포함하여 테스트 샘플내의 핵산 가닥을 증폭시키는 방법.
  2. 제1항에 있어서, 핵산 가닥이 DNA 가닥이며, 제2접촉단계 전에 약 60℃이상에서 그 핵산 가닥 및 프라이머를 60℃이상에서 DNA 중합효소 활성을 가지는 효소와 접촉시키는 것을 특징으로 하는 방법.
  3. 제1항에 있어서, 제2접촉단계를 역전사효소의 존재하에 약 42℃이상에서 수행하는 것을 특징으로 하는 방법.
  4. 제1항에 있어서, 핵산 및 프라이머를 가열하여 95℃ 이상으로 만든 후에, 상기 제2접촉단계를 수행하는 것을 특징으로 하는 방법.
  5. 제1항 내지 4항중 어느 한 항에 있어서, 제1접촉단계에서 4개의 프라이머를 사용하는 것을 특징으로 하는 방법.
  6. 제1항에 있어서, 하나의 프라이머를 다른 하나의 프라이머와 다른 농도로 제공하는 것을 특징으로 하는 방법.
  7. 제5항에 있어서, 하나의 프라이머를 다른 하나의 프라이머와 다른 농도로 제공하는 것을 특징으로 하는 방법.
  8. 제5항에 있어서, 2개의 프라이머를 다른 프라이머들과 다른 농도로 제공하는 것을 특징으로 하는 방법.
  9. 제1항에 있어서, 2개의 프라이머가 플러스-센서 프라이머이며, 내부 플러스-센스 프라이머는 프로모터-프라이머인 것을 특징으로 하는 방법.
  10. 제1항에 있어서, 2개의 프라이머가 마이너스-센스 프라이머이며, 외부 프라이머는 프로모터-프라이머인 것을 특징으로 하는 방법.
  11. 제1항에 있어서, 모든 효소 활성이 역전사효소 및 RNA 중합효소에 의해 제공되는 것을 특징으로 하는 방법.
  12. 제11항에 있어서, 효소활성이 DNA 중합효소 활성을 전혀 포함하지 않는 RNAse H에 의해 보충 되는 것을 특징으로 하는 방법.
  13. 제2항에 있어서, DNA 중합효소의 5'→3'엑소누클레이즈 활성이 결핍된 것을 특징으로 하는 방법.
  14. 제13항에 있어서, DNA 중합효소가 DNA 중합효소로부터 유도되며, 천연 형태의 DNA 중합효소는 5'→3'엑소누클레이즈 활성을 가지는 것을 특징으로 하는 방법.
  15. 제13항 또는 제14항에 있어서, DNA 중합효소가 Bacillus 종의 DNA 중합효소인 것을 특징으로 하는 방법.
  16. 제15항에 있어서, Bacillus 종이 Bacillus stearothermophilus 또는 Bacillus caldotenax인 것을 특징으로 하는 방법.
  17. 제1항에 있어서, 2개의 외부 프라이머가 2000염기 이하의 거리를 두고 핵산 가닥 또는 그것의 보체에 혼성화되는 것을 특징으로 하는 방법.
  18. 제1항에 있어서, 2개의 외부 프라이머가 500염기 이하의 거리를 두고 핵산 가닥 또는 그것의 보체에 혼성화되는 것을 특징으로 하는 방법.
  19. 제1항에 있어서, 2개의 외부 프라이머가 350염기 이하의 거리를 두고 핵산 가닥 또는 그것의 보체에 혼성화되는 것을 특징으로 하는 방법.
  20. 제6항에 있어서, 하나의 프라이머를 1내지 10μM의 농도로 제공하고 다른 프라이머를 10 내지 50μM의 농도로 제공하는 것을 특징으로 하는 방법.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019960700342A 1993-07-23 1994-07-20 핵산 증폭을 강화하는 방법 KR100189229B1 (ko)

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US9726293A 1993-07-23 1993-07-23
US8/097262 1993-07-23
US08/097262 1993-07-23
PCT/US1994/008307 WO1995003430A1 (en) 1993-07-23 1994-07-20 Methods for enhancing nucleic acid amplification

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US (1) US5786183A (ko)
EP (1) EP0656425B1 (ko)
JP (1) JP3026843B2 (ko)
KR (1) KR100189229B1 (ko)
AT (1) ATE205543T1 (ko)
AU (1) AU689789B2 (ko)
CA (1) CA2167838C (ko)
DE (1) DE69428252T2 (ko)
WO (1) WO1995003430A1 (ko)

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US5786183A (en) 1998-07-28
AU689789B2 (en) 1998-04-09
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DE69428252D1 (de) 2001-10-18
DE69428252T2 (de) 2002-04-18
KR100189229B1 (ko) 1999-06-01
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CA2167838A1 (en) 1995-02-02
EP0656425B1 (en) 2001-09-12
JP3026843B2 (ja) 2000-03-27
AU7371494A (en) 1995-02-20
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ATE205543T1 (de) 2001-09-15

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