KR960017858A - 형광 표지된 올리고뉴클레오타이드 프로브의 용액중에서의 켄칭 방법 - Google Patents

형광 표지된 올리고뉴클레오타이드 프로브의 용액중에서의 켄칭 방법 Download PDF

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KR960017858A
KR960017858A KR1019950043097A KR19950043097A KR960017858A KR 960017858 A KR960017858 A KR 960017858A KR 1019950043097 A KR1019950043097 A KR 1019950043097A KR 19950043097 A KR19950043097 A KR 19950043097A KR 960017858 A KR960017858 A KR 960017858A
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oligonucleotide
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light emission
nucleic acid
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킴 후프 피콘 테레사
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프리돌린 클라우스너 · 롤란트 보러
에프. 호프만-라 로슈 에이지
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
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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
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • 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

Abstract

본 발명은, 라벨과 상호작용하여(이때 상호작용 정도는 올리고뉴클레오타이드의 길이에 의존한다) 라벨의 광 방출을 변경시키는 DNA결합 화합물의 존재하에 광 방출을 측정함으로써, 광 방출 라벨과 표지된 올리고뉴클레오타이드 길이 변화를 검출하는 방법을 제공한다. 본 방법은 일반적으로 하이브리드화된 올리고뉴클레오타이드 프로브의 선택적 절단을 야기하는 반응을 이용하는 핵산 서열 검출 검정 및, 특정하게는 하이브리드화된 프로브가 프라이머 연장과 동시에 절단되는 증폭/검출 검정에 관한 것이기도 하다.

Description

형광 표지된 올리고뉴클레오타이드 프로브의 용액중에서의 켄칭 방법
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 실시예 1에 기술된 바와 같은, 표지된 올리고뉴클레오타이드 길이, 광 방출 라벨 및 DNA결합 화합물의 농도에 따른 켄칭 의존성을 나타낸 그래프이다.
제2도는 실시예 2에 기술된 바와 같은, 사용된 폴리에틸렌아민(PEI) 농도 및 분자량에 따른 켄칭 의존성을 나타내는 그래프이다.
제3도는 실시예 3에 기술된 바와 같은, 내부적인 대조표준의 존재하에 표적 핵산 서열 증폭에 의한 핵산을 정량화 그래프이다.

Claims (6)

  1. (a) 올리고뉴클레오타이드의 길이를 변환시키는 조건하에서 반응 혼합물 중에서 반응을 수행하는 단계; (b) 라벨과 상호작용함으로써 (이때 상호작용은 올리고 뉴클레오타이드의 길이에 의존한다) 라벨의 광 방출을 변경시킬 수 있는 DNA결합 화합물의 존재하에 반응 혼합물 중의 라벨의 광 방출을 측정하는 단계; 및 (c) 단계(b)에서 측정된 광 방출에 의해 올리고뉴클레오타이드의 길이 변화를 검출하는 단계들을 포함하는, 광방출라벨로 표지된 올리고뉴클레오타이드의 길이 변화를 검출하는 방법.
  2. 제1항에 있어서, (a1) 표적 핵산과 하이브리드화할 수 있는 서열을 함유하고 광 방출 라벨로 표지된 일본 쇄 올리고뉴클레오타이드 및 샘플을 포함하는, 특정 반응용 반응기 혼합물(여기서, 반응은 올리고뉴클레오타이드가 표적 핵산에 하이브리드화하는 경우에만 올리고뉴클레오타이의 절단을 촉매화한다)을 제공하는 단계; (a2) 올리고뉴클레이오타이드가 표적 서열과 하이브리드화 되어 절단되는 조건하에서 상기 혼합물을 처리하는 단계; (b) 라벨의 광 방출을 변경시킬 수 있는 DNA결합 화합물의 존재하에 반응 혼합물 중 라벨의 광 방출을 측정하는 단계(이때, 상호작용은 올리고뉴클레오타이드의 길이에 의존한다); 및 (c) 단계(b)에서 측정된 광방출에 의해 표적 서열의 존재 여부를 결정하는 단계들을 포함하는, 샘플 중의 표적핵산을 검출하기 위한 방법.
  3. 제2항에 있어서, 표적 서열을 단계(b) 이전에 증폭시키는 방법.
  4. 제3항에 있어서, (a1) 샘플, 한쌍의 올리고뉴클레오타이드 프라이머, 5′→3′ 뉴클레아제 활성을 갖는 핵산 폴레머라제 및 표적 핵산의 영역에 하이브리드화할 수 있는 올리고뉴클레오타이드[여기서, 올리고뉴클레오타이드는 올리고뉴클레오타이드 프라이머에 의해 결합되는 표적 핵산 서열 내에서 하이브리드화되고, 올리고 뉴크레오타이드 프로브는 광 방출 라벨로 표지된다]를 포함하는 폴리머라제 연쇄 반응(PCR) 혼합물을 제공하는 단계; (a2) 5′→3′ 뉴클레아제 h할성을 갖는 핵산 폴리머라제가 표적서열과 하이브리드화된 올리고뉴클레오타이드를 절단하도록 PCR조건하에서 PCR 혼합물을 처리하는 단계; (b) 라벨의 광 방출을 변경시킬 수 있는 DNA결합 화합물의 존재하에 반응 혼합물 중의 라벨의 광 방출을 측정하는 단계(이때, 상호작용은 올리고뉴클레오타이드의 길이에 의존한다); 및 (c) 단계(b)에서 측정되는 광 방출에 의해 표적 서열의 존재 여부를 결정하는 단계들을 포함하는 방법.
  5. 제1항 내지 제4항 중 어느 한 항에 있어서, DNA결합 화합물이 폴리에틸렌아민 또는 이의 유도체인 방법.
  6. 제1항 내지 제5항 중 어느 한 항에 있어서, 라벨이 플루오레스세인, 로다민 및 이의 유도체로 이루어진 그룹중에서 선택되는 방법.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019950043097A 1994-11-23 1995-11-23 형광표지된올리고뉴클레오타이드프로브의용액중에서의켄칭방법 KR100431421B1 (ko)

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US08/347657 1994-11-23
US08/347,657 US5571673A (en) 1994-11-23 1994-11-23 Methods for in-solution quenching of fluorescently labeled oligonucleotide probes
US08/347,657 1994-11-23

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KR100431421B1 KR100431421B1 (ko) 2004-12-03

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CN (1) CN1131320C (ko)
AT (1) ATE204333T1 (ko)
AU (1) AU700885B2 (ko)
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DE (1) DE69522176T2 (ko)
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