KR920702866A - 핵산 증폭 방법 - Google Patents

핵산 증폭 방법

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KR920702866A
KR920702866A KR1019910700397A KR910700397A KR920702866A KR 920702866 A KR920702866 A KR 920702866A KR 1019910700397 A KR1019910700397 A KR 1019910700397A KR 910700397 A KR910700397 A KR 910700397A KR 920702866 A KR920702866 A KR 920702866A
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dna
primer
sequence
rna
polymerase
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티. 말레크 로렌스
데이비 레릴
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에프. 쥐. 엠, 헤르만스 피. 시. 샬크위크
악조 엔. 브이.
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract

내용 없음

Description

핵산 증폭 방법
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
본 발명의 구체예를 설명하는 도면에서
제1도는 핵산 증폭방법을 나타낸 일반적 도식이다.
제2도는 증폭 방법 실험에 사용하는 합성 뉴클레오티드 DNA 서열을 나타내며, 제2A도는 개그(gag) 실험서열이고; 제2B도는 gag 2 실험 서열이다.
제3도는 상이한 프라이머 농도를 사용하는 증폭반응의 PAGE 분서결과의 방사선사진이다.
제4도는 상이한 주형 농도를 사용하는 증폭 반응의 PAGE 분석결과의 방사선사진이다.
제5도는 증폭 반응에서 롯트-블롯(Dot-blot) 하이브리드와의 방사선사진이다.
제6도는 주형으로 제한 단편물을 사용한 증폭 반응의 PAGE 분석결과의 방사선사진이다.
제7도는 비지시성 핵산 증폭 방법에 관한 일반적 도식이다.

Claims (64)

  1. 단일쇄 RNA, 단일쇄 DNA와 이중쇄 DNA를 합성하는 것을 포함하는 특정한 핵산서열의 증폭방법에 있어서, 상기 단일쇄 RNA는 제1 프라이머를 위한 제1 주형이며, 상기 단일쇄 DNA는 제2 프라이머를 위한 제2 주형이며, 상기 이중쇄 DNA는 상기 제1 주형의 복사체를 다수 합성하기 위한 제3 주형이며, 상기 제1 프라이머 또는 제2 프라이머의 서열은 상기 특정 핵산 서열의 서열에 대한 충분한 상보성이 있으며, 상기 제1 프라이머 또는 제2 프라이머의 서열은 상기 특정 핵산 서열의 서열에 대한 충분한 상동성이 있으며, 상기 제1 프라이머의 3' 말단은 상보쇄 상의 상기 제2 프라이머의 3' 말단을 향해 배향되는 것을 특징으로 하는 특정한 핵산서열의 증폭방법.
  2. 제1항에 있어서, 상기 제1프라이머는 상기 제1 주형에 선택적으로 하이브리드하며, 상기 제1프라이머에 공유결합되고 상기 제1 주형에 대한 상보성이 있는 DNA의 합성을 촉진하며; RNA폴리머라제에 대한 전사개시 부위 서열과 프로모터 서열을 갖고 있는 제2 프라이머는 제2 주형에 선택적으로 하이브리드 함으로써, 상기 제2프라이머에 공유결합되고 상기 제2 주형에 대한 상보성이 있는 DNA를 합성하는 것을 특징으로 하는 방법.
  3. (a) DNA의 제1프라이머를 제1 주형에 하이브리드 시키고, 상기 제1프라이머는 상기 제1 주형의 RNA 서열에 대해 충분한 상보성이 있는 서열을 갖고 있으며; (b) 상기 제1프라이머에 공유결합되며, 상기 제1 주형의 RNA 서열에 대해 충분한 상보성이 있는 제1 DNA서열을 합성하고, 여기에서 상기 제1 DNA서열 및 제1 프라이머는 제2 주형을 포함하며; (c) 제2프라이머를 하이브리드화 하기 위해 상기 제2주형으로부터 상기 제1주형을 분리하고; (d) 상기 제2 주형에 DNA의 제2프라이머를 하이브리드시키고, 여기에서 상기 제2프라이머는 상기 제2 주형의 DNA 서열에 대해 충분히 상보성이 있는 서열을 가지며, 상기 제2 프라이머는 RNA폴리머라제에 대한 프로모터 서열 및 전사개시 부위 서열을 갖으며; (e) 상기 제2프라이머에 공유결합되며, 상기 제2 주형의 DNA 서열에 대해 상보성이 있는 제2 DNA서열을 합성하고, 상기 제2 주형에 공유결합되고, 상기 제2 프라이머의 DNA 서열에 대해 상보성이 있는 제3 DNA서열을 합성하고 여기에서 상기 제2 및 제3 DNA 서열, 상기 제2 프라이머와 상기 제2 주형은 제3주형을 포함하며; (f) 상기 제3 주형으로부터 상기 제1 주형의 RNA 서열 복사체를 다수 합성하는 것을 포함하고, 상기 제1 또는 제2 프라이머의 서열은 상기 특정 핵산 서열의 서열에 대한 충분한 상보성이 있으며, 상기 제1 또는 제2 프라이머는 상기 특정 핵산 서열의 서열에 대해 충분한 상동성이 있으며, 상기 제1 프라이머의 3' 말단은 상보쇄 상의 상기 제2 프라이머의 3' 말단을 향해 배향되는 특정 핵산서열의 증폭방법.
  4. 제3항에 있어서, 상기 제2프라이머는 그 3' 말단에 상기 제2 주형의 DNA 서열에 대해 충분한 상보성이 있는 서열을 갖으며, 그 5' 말단에는 폴리머라제에 대한 전사개시 부위 서열과 프로모터 RNA 서열을 갖는 방법.
  5. 제4항에 있어서, 상기 제2 주형에 공유결합된 상기 제3 DNA 서열이 상기 제2프라이머의 5' 말단에 있는 RNA 서열에 대해 상보성이 있는 방법.
  6. 제3항에 있어서, 제1 DNA 서열은 RNA의 존성 DNA 폴리머라제에 의해 합성되는 방법.
  7. 제3항에 있어서, 상기 제1주형은 RNA-DNA 하이브리드에 대해 특이성이 있는 리보 뉴클레아제에 의해 제2주형으로부터 분리되는 방법.
  8. 제7항에 있어서, 상기 리보 뉴클레아제는 E. coli 리보뉴클레아제 H인 방법.
  9. 제7항에 있어서, 상기 리보 뉴클레아제는 송아지 흉선 리보뉴클레아제 H인 방법.
  10. 제3항에 있어서, 상기 제2 및 제3 DNA서열이 DNA 의존성 DNA 폴리머라제로 합성되는 방법.
  11. 제3항에 있어서, 상기 제1 주형은 DNA 의존성 RNA 폴리머라제에 의해 상기 제3 주형으로부터 합성되는 방법.
  12. 제3항에 있어서, 상기 제1프라이머는 프로모터 서열 및 전사개시 부위 서열을 갖는 방법.
  13. 제3항에 있어서, 상기 제1프라이머는 지지체 수단에 하이브리드 되는 서열을 갖는 방법.
  14. 제3항에 있어서, 상기 제2프라이머는 그 3'말단으로부터 그 5'말단쪽으로 충분히 상보성 있는 서열, 전사개시 부위 서열 및 프로모터 서열을 갖는 방법.
  15. 제14항에 있어서, 상기 전사개시 부위 서열 및 프로모터 서열이 박테리오 파아지 RNA 폴리머라제와 결합하는 방법.
  16. 제14항에 있어서, 상기 전사개시 부위 서열 및 프로모터 서열은 T7 RNA 폴리머라제와 결합하는 방법.
  17. 제14항에 있어서, 상기 전자개시 부위 서열 및 프로모터 서열은 AATTCTAATAC-GACTCACTATAGGGAG인 방법.
  18. 제11항에 있어서, 상기 DNA 의존성 RNA 폴리머라제는 T7 RNA 폴리머라제인 방법.
  19. 제11항에 있어서, 상기 DNA 의존성 RNA 폴리머라제는 박테리오 파아지 RNA 폴리머라제인 방법.
  20. 제11항에 있어서, 상기 DNA 의존성 RNA 폴리머라제는 파이지 T3 폴리머라제인 방법.
  21. 제11항에 있어서, 상기 DNA 의존성 RNA 폴리머라제는 파이지 ΦⅡ 폴리머라제인 방법.
  22. 제11항에 있어서, 상기 DNA 의존성 RNA 폴리머라제는 파이지 sp6 폴리머라제인 방법.
  23. 제11항에 있어서, 상기 DNA 의존성 RNA 폴리머라제는 슈도모나스 파아지 gh-1 폴리머라제인 방법.
  24. 제6항에 있어서, 상기 RNA 의존성 DNA 폴리머라제는 레트로바이러스 폴리머라제인 방법.
  25. 제24항에 있어서, 상기 레트로바이러스 폴리머라제는 조류의 근원세포중 바이러스 폴리머라제인 방법.
  26. 제24항에 있어서, 상기 레트로바이러스 폴리머라제는 말로니(Maloney) 쥐의 백혈병 바이러스 폴리머라제인 방법.
  27. 제6항, 제10항 또는 제11항에 있어서, 상기 폴리머라제는 엑소뉴클레아제 또는 엔도뉴클레제 활성이 부족한 것을 특징으로 하는 방법.
  28. 제10항에 있어서, 상기 DNA 의존성 DNA 폴리머라제는 조류의 근원세포중 바이러스 폴리머라제인 방법.
  29. 제1프라이머, 제2프라이머, 리보뉴클레아제 H, RNA 의존성 DNA 폴리머라제, DNA 의존성 DNA 폴리머라제, RNA 폴리머라제, 리보뉴클레오시드 트리포스페아트(NTP)와 데옥시리보뉴클레오시드 트리포스페이트(dNTP)를 샘플과 혼합하는 단계를 포함하고, 상기 DNA의 제1 프라이머는 RNA의 제1 주형에 대해 충분한 상보성이 있는 서열을 갖으며; 상기 DNA의 제2 프라이머는 DNA의 제2 주형에 대해 충분한 상보성이 있는 서열, 프로모터의 서열, RNA 폴리머라제에 대한 기질로서 인식되는 전사 개시 부위 서열을 지니며; 상기 제1 또는 제2 프라이머의 서열은 상기 특정 핵산 서열의 서열에 대해 충분한 성보성이 있으며, 상기 제1 또는 제2 프라이머의 서열은 상기 특정 핵산 서열에 대한 충분한 상동성이 있으며, 상기 제1 프라이머의 3' 말단은 상보쇄 상의 상기 제2 프라이머의 3' 말단을 향해 배양되는 있는 특정 핵산서열의 증폭방법.
  30. 제1프라이머, 제2프라이머, 조류의 근원세포중 바이러스 폴리머라제, E. coli 리보뉴클레아제 H, 박테리어 파아지 T7 RNA 폴리머라제, NTP와 dNTP를 샘플과 혼합하는 단계를 포함하고, 상기 제1 프라이머는 RNA의 제1 주형에 대해 충분한 상보성이 있는 서열을 갖으며; 상기 제2 프라이머는 ONA의 제2 주형에 대해 충분한 상보성이 있는 서열, T7 RNA 폴리머라제에 대한 기질로서 인식되는 전사 개시 부위 서열 및 프로모터 서열을 갖으며; 상기 제1 프라이머 또는 제2 프라이머의 서열은 상기 특정 핵산 서열의 서열에 대해 충분한 상보성이 있으며, 상기 제1 프라이머 또는 제2 프라이머의 서열은 상기 특정 핵산 서열의 서열에 대한 충분한 상동성이 있으며, 상기 제1 프라이머의 3' 말단은 상보쇄 상의 제2 프라이머의 3' 말단을 향해 배양되는 있는 특정 핵산서열의 증폭방법.
  31. 제1항 또는 제3항에 있어서, 상기 특정 핵산 서열을 포함하고 있는 것으로 예상되는 제1샘플의 증폭량과 상기 핵산 서열이 존재하고 있지 않은 제2샘플의 증폭량을 비교함으로써 상기 특정한 핵산서열의 존재를 검출하는 것을 추가로 포함하는 방법.
  32. 제29항 또는 제30항에 있어서, 상기 특정한 핵산 서열을 포함하고 있는 것으로 예상되는 제1샘플의 증폭량과 상기 핵산 서열이 존재하고 있지 않은 제2샘플의 증폭량을 비교함으로써 상기 특정한 핵산서열의 존재를 검출하는 것을 추가로 포함하는 방법.
  33. 제1항 또는 제3항에 있어서, 상기 특정 핵산 서열 또는 이 특정한 핵산 서열의 생성물에 하이브리드하는 프로오브를 사용하여 상기 특정한 핵산 서열의 존재를 검출하는 것을 추가로 포함하는 방법.
  34. 제1항 또는 제3항에 있어서, 제한 엔도뉴클레아제와 전기영동 분리법을 사용하여 상기 특정한 핵산 서열의 존재를 검출하는 것을 추가로 포함하는 방법.
  35. 제31항 또는 제32항에 있어서, 제한 엔도뉴클레아제와 전기영동 분리법을 사용하여 상기 특정한 핵산 서열의 존재를 검출하는 것을 추가로 포함하는 방법.
  36. 제31항 또는 제32항에 있어서, 특정한 핵산 서열 또는 그것의 생성물에 하이브리드하는 프로브를 갖는 특정한 핵산 서열의 존재를 검출하는 것을 추가로 포함하는 방법.
  37. 제1항의 방법에 의해 증폭된 특정한 핵산 서열.
  38. 제3항의 방법에 의해 증폭된 특정한 핵산 서열.
  39. 제29항의 방법에 의해 증폭된 특정한 핵산 서열.
  40. 제30항의 방법에 의해 증폭된 특정한 핵산 서열.
  41. (a) 제1 주형의 RNA 서열에 대해 충분히 상보성이 있는 DNA서열을 갖는 제1 프라이머; (b) 제2 주형의 DNA 서열에 대해 상보적인 DNA서열을 갖는 제2 프라이머; (c) 리보뉴클레아제 H; (d) RNA-의존성 DNA 폴리머라제; (e) DNA-의존성 RNA 폴리머라제; (f) DNA-의존성 DNA 폴리머라제; (g) NTP(리보뉴크레오사이드 트리포스페이트); 및 (h) dNTP(데옥시리보뉴클레오시드 트리포스페아트)를 포함하며, 상기 제1 프라이머 또는 제2 프라이머의 서열은 특정한 핵산 서열과 충분히 상보성이고, 상기 제1 프라이머 또는 제2 프라이머의 서열은 상기 특정한 핵산 서열과 충분히 상동하며, 제1 프라이머의 3'-말단은 상보성 가닥상의 제2 프라이머의 3'-말단쪽으로 배양되어 있는 것을 특징으로 하는 특정한 핵산서열의 증폭을 위한 키트.
  42. (a) 제1 주형의 RNA 서열에 충분히 상보적인 DNA서열을 갖는 제1 프라이머; (b) 제2 주형의 DNA 서열에 상보적인 DNA서열을 갖는 제2 프라이머; (c) 박테리오파지 T7 RNA 폴리머라제; (d) 이. 콜리 리보뉴클레아제 H; (e) 조류 근원세포증(myoblastosis) 바이러스의 폴리머라제; (f) NTP; 및 (g) dNTP를 포함하며, 상기 제1 프라이머 또는 제2 프라이머의 서열은 상기 특정한 핵산 서열과 충분히 상보적이고, 제1 프라이머 또는 제2 프라이머의 서열은 특정한 핵산 서열과 충분히 상동하며, 제1 프라이머의 3'-말단은 상보성 가닥상의 제2 프라이머의 3'-말단쪽으로 배향되어 있는 것을 특징으로 하는 특정한 핵산 서열의 증폭하기 위한 키트.
  43. (A) 적어도 하나의 프라이머가 기능성 프로모터의 서열을 포함하는 제1올리고뉴클레오티드 프라이머와 제2올리고뉴클레오티드 프라이머; RNA-의존성 DNA 폴리머라제, DNA-의존성 DNA 폴리머라제, DNA-의존성 RNA 폴리머라제; 단일쇄나 이중쇄 RNA 또는 DNA에 결합되지 않는 RNA/DNA 하이브리드의 DNA를 방출할 수 있는 리보뉴클레아제; 및 NTP와 dNTP를 포함하는 단일 반응 배지를 제공하는 단계 (B) 하기 ⅰ) 내지 ⅲ) 중 하나 이상을 상기 반응 배지에 첨가하는 단계; ⅰ) RNA분자; ⅱ) a) 상기 제1올리고뉴클레오티드 프라이머의 3'-말단 부위에 하이 브리드되는 3'-말단서열; 또는 b) 기능성 프로모터의 서열인 5'-말단서열; 또는 c) 상기 제2올리고뉴클레오티드 프라이머의 3'-말단 부위에 하이 브리드되는 3'-말단서열을 포함하는 단일쇄 DNA 분자, ⅲ) a) (B)의 ⅱ)에 기술된 적어도 하나의 단일쇄 DNA 또는 b) 기능성 프로모터가 증폭부위에 인접해 있고 상기 부위의 조절전사로 배향된 증폭가능 부위 및 기능성 프로모터, (C) 매우 일정한 온도에서 반응 사이클이 상기 반응 배지중에서 개시되고, 상기 사이클은 하기한 제1반응 셋트와 제2반응 셋트중 적어도 하나를 포함하는 단계 : 제1반응 셋트는 ⅰ) 상기 제1올리고뉴클레오티드 프라이머가 RNA 분자의 3'말단 부위에서 하이브리드되며, RNA/DNA 하이브리드는 상기 RNA-의존성 DNA 폴리머라제의 작용에 의해 RNA/DNA 하이브리드를 형성하고, 상기 RNA/DNA 하이브리드는 제1 DNA 단편을 포함하며, 상기 제1 DNA 단편은 상기 제1올리고뉴클레오티드 프라이머에 공유결합하고 상기 RNA 분자의 적어도 일부분에 상보적이며; ⅱ) 상기 리보뉴클레아제는 상기 RNA/DNA 하이브리드에 작용하여 적어도 몇몇의 상기 RNA 분자를 절단하고, 상기 제1 DNA 단편을 상기 RNA/DNA 하이브리드에서 분리해내며, 상기 제1 DNA 단편은 상기 제2올리고뉴클레오티드 프라이머와 하이브리드하여 상기 DNA-의존성 DNA 폴리머라제에 의해 작용되는 이중나선을 형성함으로써 a) 제2올리고뉴클레오티드 프라이머에 공유결합하고 상기 제1 DNA 단편의 적어도 일부분에 상보적인 제2 DNA 단편과 b) 상기 제1 DNA 단편에 공유결합하고 제2프라이머의 적어도 일부분에 상보적인 제3 DNA 단편을 생성하고; 및 ⅲ) 상기 RNA 폴리머라제가 (C)의 ⅰ)의 RNA 분자에 상응하는 다수의 RNA 분자를 생성하고, 제2반응 셋트는 ⅰ') 상기 제2올리고뉴클레오티드 프라이머가 RNA 분자의 3'말단 부위에서 하이브리드되며, RNA/DNA 하이브리드는 상기 RNA-의존성 DNA 폴리머라제의 작용에 의해 RNA/DNA 하이브리드는 제2 DNA 단편을 포함하며, 상기 제2 DNA 단편은 상기 제2올리고뉴클레오티드 프라이머에 공유결합하고 상기분자의 적어도 일부분에 상보적이며; ⅱ') 상기 리보뉴클레아제는 상기 RNA/DNA 하이브리드에 작용하여 적어도 몇몇의 상기 RNA 분자를 절단하고, 상기 제2 DNA 단편을 상기 RNA/DNA 하이브리드에서 분리해내며, 상기 제2 DNA 단편은 제1올리고뉴클레오티드 프라이머와 하이브리드하여 상기 DNA-의존성 DNA 폴리머라제에 의해 작용되는 이중나선을 형성함으로써 제1올리고뉴클레오티드 프라이머에 공유결합하고 상기 제2 DNA 단편의 적어도 일부분에 상보적인 제1 DNA 단편을 생성하고; 및 ⅲ') 상기 RNA 폴리머라제가 (C)의 ⅰ)의 RNA 분자에 상보적인 분자에 사용하는 다수의 RNA 분자를 생성하며, (D) 그후 상기 (C)의 ⅲ)또는 (C)의 (ⅲ')의 RNA분자, 상기 제1 DNA 단편, 상기 제2 DNA 단편 및 상기 2중쇄 DNA로 구성된 그룹 중 적어도 하나가 예정 농도로 검출될때까지 상기 조건을 유지키기는 단계들을 포함한, 연속적인 시약첨가 도는 온도 재순환 없이 핵산 서열을 증폭시키는 방법.
  44. 제43항에 있어서, 단계(B)가 단계(C) ⅰ)의 RNA 분자에 안티센스(antisense)한 RNA 분자를 첨가하는 것을 포함하는 방법.
  45. 제43항에 있어서, 단계(B)가 단계(C) ⅰ')의 RNA 분자에 안티센스한 RNA 분자를 첨가하는 것을 포함하는 방법.
  46. 제43항에 있어서, 단계(B)가 상기 반응배지에 RNA 분자를 첨가하는 것을 포함하는 방법.
  47. 제43항에 있어서, 단계(B)가 상기 반응배지에 단일쇄 DNA 분자를 첨가하는 것을 포함하는 방법.
  48. 제43항에 있어서, 단계(B)가 상기 반응배지에 이중쇄 DNA 분자를 첨가하는 것을 포함하는 방법.
  49. 제43항에 있어서, 단계(C)가 제1 반응셋트를 포함하는 방법.
  50. 제43항에 있어서, 단계(C)가 제2 반응셋트를 포함하는 방법.
  51. 제43항에 있어서, 단계(D)가 단계(C) ⅲ)의 RNA 분자의 농도를 모니터하는 것을 포함하는 방법.
  52. 제43항에 있어서, 단계(D)가 단계(C) ⅲ')의 RNA 분자의 농도를 모니터하는 것을 포함하는 방법.
  53. 제43항에 있어서, 단계(D)가 제1 DNA 단편의 농도를 모니터하는 것을 포함하는 방법.
  54. 제43항에 있어서, 단계(D)가 제2 DNA 단편의 농도를 모니터하는 것을 포함하는 방법.
  55. 제43항에 있어서, 단계(D)가 제2중쇄 DNA 의 농도를 모니터하는 것을 포함하는 방법.
  56. 제43항에 있어서, 단계(D)가 30분 내지 4시간 동안 상기 조건을 유지하는 것을 포함하는 방법.
  57. 제43항에 있어서, 전사가 개시될 수 있도록 RNA 폴리머라제가 상기 전사 개시부위의 서열과 상기 기능성 프로모터의 서열을 인지하는 방법.
  58. 제43항에 있어서, 상기 DNA-의존성 DNA 폴리머라제가 DNA 폴리머라제 α또는 S인 방법.
  59. 제43항에 있어서, 상기 DNA-의존성 DNA 폴리머라제가 송아지 흉선 DNA 폴리머라제인 방법.
  60. 제43항에 있어서, 상기 DNA-의존성 DNA 폴리머라제가 DNA 폴리머라제 결핍성 엑소 뉴클레아제 활성을 갖는 방법.
  61. 제43항의 방법에 의해 증폭된 핵산 서열을 포함한 RNA 분자 또는 DNA 분자.
  62. 제43항에 있어서, 상기 증폭된 핵산을 클로닝 벡터에 결착시키고 그후 상기 핵산 서열을 클로닝하거나 형질 발현 시스템에서 상기 핵산 서열에 의해 코오딩된 생성물을 형질발현시키는 단계를 추가로 포함하는 방법.
  63. (a) 제1올리고뉴클레오티드 프라이머 용액을 함유한 저장소, (b) 제2올리고뉴클레오티드 프라이머용액을 함유한 저장소, (c) 단일쇄 또는 이중쇄 RNA나 DNA가 결합되지 않은 RNA/DNA 하이브리드의 RNA를 가수분해할 수 있는 리보뉴클레아제용액을 함유한 저장소, (d) RNA-의존성 RNA 폴리머라제용액을 함유한 저장소, (e) DNA-의존성 RNA 폴리머라제용액을 함유한 저장소, (f) DNA-의존성 DNA 폴리머라제 용액을 함유한 저장소, (g) NTP용액을 함유한 저장소, 및 (h) dNTP용액을 함유한 저장소들의 어셈블리를 함유한 핵산 분자를 증폭시키기 위한 키트.
  64. 기능성 프로모터의 단일쇄 서열을 포함하는 제2올리고뉴클레오티드 프라이머와 제1올리고뉴클레오티드 프라이머; RNA-의존성 DNA 폴리머라제; DNA-의존성 DNA 폴리머라제; DNA-의존성 RNA 폴리머라제; dNTP와 NTP에 결합되지 않은 RNA/DNA 하이브리드의 DNA를 방출할 수 있는 리보뉴클레아제를 포함하는 단일 반응 배지를 제공하는 단계 (B) 하기 ⅰ) 내지 ⅲ) 중 하나 이상을 상기 반응 배지에 첨가하는 단계; ⅰ) RNA 분자, ⅱ) a) 상기 제1올리고뉴클레오티드 프라이머의 3'-말단 부위에 하이 브리드되는 3'-말단서열; 또는 b) 기능성 프로모터의 단일쇄 서열인 5'-말단서열; 또는 c) 상기 제2올리고뉴클레오티드 프라이머의 3'-말단 부위에 하이브리드되는 3'-말단서열을 포함하는 단일쇄 분자, 또는 ⅲ) 기능성 프로모터는 증폭부위에 인접해 있고 상기 부위의 조절전사로 배향된 증폭가능 부위 및 기능성 프로모터를 함유한 이중쇄 DNA분자, (C) 다수의 반응 사이클이 상기 반응 배지중에서 개시될 수 있도록 적어도 한기한 단계들을 포함하는 조건을 설정하는 단계: ⅰ) 상기 제1올리고뉴클레오티드 프라이머가 RNA 분자의 3'말단 부위에서 하이브리드되며, RNA/DNA 하이브리드는 상기 RNA-의존성 DNA 폴리머라제의 작용에 의해 RNA/DNA 하이브리드를 형성하고, 상기 RNA/DNA 하이브리드는 제1 DNA 단편을 포함하며, 상기 제1 DNA 단편은 상기 제1올리고뉴클레오티드 프라이머에 공유결합하고 상기 RNA 분자의 적어도 일부분에 상보적인 단계이며; ⅱ) 상기 리보뉴클레아제가 상기 RNA/DNA 하이브리드에 작용하여 적어도 몇몇의 상기 RNA 분자를 절단하고, 상기 제1 DNA 단편을 상기 RNA/DNA 하이브리드에서 분리해내며, 상기 제1 DNA 단편은 그것의 3'-말단에서 제2올리고뉴클레오티드 프라이머와 하이브리드하여 상기 DNA-의존성 DNA 폴리머라제에 의해 작용되는 이중나선을 형성함으로써 a) 제2올리고뉴클레오티드 프라이머에 공유결합하고 상기 제1 DNA 단편의 적어도 일부에 상보적인 제2 DNA 단편과 b) 상기 제1 DNA 단편에 공유결합하고 제2프라이머의 적어도 일부분에 상보적인 제3 DNA 단편을 생성하고; 및 ⅲ) 상기 RNA 폴리머라제가 (C)의 ⅰ)의 RNA 분자와 상동한 다수의 RNA 분자를 생성하는 단계들을 포함하는 것을 특징으로 하는 매우 일정한 온도에서 핵산 서열을 증폭시키는 방법.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019910700397A 1988-02-24 1989-08-19 핵산 증폭 방법 KR920702866A (ko)

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ATE106948T1 (de) 1994-06-15
WO1991002814A1 (en) 1991-03-07
EP0329822B1 (en) 1994-06-08
EP0329822A3 (en) 1990-08-01
US5409818A (en) 1995-04-25
ES2053648T3 (es) 1994-08-01
DE3850093D1 (de) 1994-07-14
CA1340807C (en) 1999-11-02
KR960015744B1 (ko) 1996-11-20
KR890013184A (ko) 1989-09-21
EP0329822A2 (en) 1989-08-30

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