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KR890014750A - 뉴클레오티드 서열을 검출하는 방법 - Google Patents

뉴클레오티드 서열을 검출하는 방법 Download PDF

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KR890014750A
KR890014750A KR1019890003001A KR890003001A KR890014750A KR 890014750 A KR890014750 A KR 890014750A KR 1019890003001 A KR1019890003001 A KR 1019890003001A KR 890003001 A KR890003001 A KR 890003001A KR 890014750 A KR890014750 A KR 890014750A
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로보트 뉴톤 클라이브
프레드 마르크함 알렉산더
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그라함 도날드 아놀드
임페리알 케미칼 인더스트리스 피엘씨
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Abstract

내용 없음.

Description

뉴클레오티드 서열을 검출하는 방법
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1(a)도 및 제1(b)도는 본 발명의 첫번째 구체예를 도시한 도면이다.
제1(c)도는 전기 영동 시험의 일반적 결과를 기록한 도면이다.
제2(a)도 및 제2(b)도는 본 발명의 두번째 구체예를 도시한 도면이다.
제3도는 다수 증폭 생성물을 구별하기 위한 바람직한 방법을 도시한 도면이다.

Claims (15)

  1. 샘플에 포함된 하나 혹은 그 이상의 핵산에 적어도 하나의 변이 뉴클레오티드의 존재 유무를 검출하는 방법에 있어서, 상기 샘플을 적절한 뉴클레오시드 트리포스페이트, 상기 뉴클오시드 트리포스페이트를 중합시키는 기능물(reagent) 및 표적 열기 서열의 진단부분에 대한 진단 프라이머를 하이 브리드화 조건하에서 함께 혹은 연속적으로 처리함으로써 상기 진단 프라이먼의 뉴클레오시드 서열이 상기 진단부분에 거의 상보적인 상태이고, 진단 프라이머의 터미날 뉴클레오티드가 예견되는 변이 뉴클레오티드 혹은 그에 상응하는 정상적인 뉴클레오티드 중 어느 하나에 상보적인 상태이어서, 진단 프라이머의 상기 터미날 뉴클레오티드가 상기 표적 염기 서열의 상응하는 뉴클레오티드에 상보적일 경우 상기 진단 프라이머의 확장생성물이 합성되고, 진단 프라이머의 상기 터미날 뉴클레오티드가 표적 염기 서열의 상응하는 뉴클레오티드와 상보적이지 않을 경우에는 어떠한 확장 생성물도 합성되지 않으며 ; 및 확장 생성물의 존재 유무로 부터 상기 예견되는 변이 뉴클레오티드의 존재 유무를 검출하는 것으로 구성되는 방법.
  2. 제 1항에 있어서, (1) 상기 샘플을 적절한 뉴클레오시드 트리포스페이트, 상기 뉴클레오시드 트리포스페이트를 중합화시키는 기능물, 표적 염기 서열의 진단부분에 대한 진단 프라이머 및 상응하는 증폭 프라이머를 하이브리드화 조건하에서 함께 혹은 연속적으로 처리하여 상기 진단 프라이머의 뉴클레오티드 서열이 상기 진단부분에 거의 상보적인 상태이고, 진단 프라이머의 터미날 뉴클레오티드는 예견되는 변이 뉴클레오티드 혹은 그에 상응하는 정상적인 뉴클레오티드 중 어느 하나에 상보적인 상태이어서 진단 프라이머의 상기 터미날 뉴클레오티드가 상기 표적 염기 서열의 상응하는 뉴클레오티드와 상보적일 경우 진단 프라이머의 확장생성물이 합성되고, 진단 프라이머의 상기 터미날 뉴클레오티드가 상기 표적 염기 서열의 상응하는 뉴클레오티드와 상보적이지 않는 경우에는 어떠한 확장 생성물도 합성되지 않으며 및 진단 프라이머의 합성된 확장생성물이 그것의 상보본쇄로 부터 분리된 후에는 상기 증폭 프라이머 확장 생성물을 합성하는 주형으로서 작용할 수 있으며 ; (2) 상기 샘플을 변성화 조건에서 처리하여 상기 프라이머 확장 생성물을 그러한 확장 생성물이 합성되어지는 그것의 주형으로 부터 분리시키고 ; (3) 단계(2)에서 생성된 일본쇄를 적절한 뉴클레오시드 트리포스페이트, 상기 뉴클레오시드 트리포스페이트를 중합화시키는 기능물, 진단 프라이머 및 본 명세서상에서 규정된 증폭 프라이머와 함께 연속적으로 접촉시키고, 단계 (2)에서 생성된 일본쇄를 주형으로서 작용함으로써 가능한 곳에서는 진전된 확장 생성물을 합성하고, (4) 단계(2) 및 (3)을 충분히 여러번 반복시켜 상기 적절한 뉴클레오시드 서열을 감지할 수 있을 정도로 증폭시키고 ; 및 (5) 단계(4)에서 수득한 증폭 생성물의 존재 유무로 부터 상기 예견되는 변이 뉴클레오티드의 존재 유무를 검출하는 것을 포함하는 방법.
  3. 제1항에 있어서, 상기 샘플을 하기(a) 또는 (b)와 함께 혹은 연속으로 처리하는데 하기의 첫번째 진단 프라이머의 터미날 뉴클레오티드 및 두번째 진단 프라이머의 터미날 뉴클레오티드는 둘다 각 프라이머의 5′ 말단 혹은 3′ 말단중 어느 하나에서 존재하고 하기 첫번째 핵산 서열은 하기 두번째 핵산 서열에 대하여 반대로 존재하는 것을 특징으로 하는 방법. (a) 첫번째 핵산 서열의 진단부분에 거의 상보적인 서열을 지니며, 상기 예견되는 변이 뉴클레오티드에 상보적인 터미날 뉴클레오티드를 가지는 첫번째 진단 프라이머, 및 두번째 핵산 서열의 진단부분에 거의 상보적인 서열을 지니며 상기 예견되는 변이 뉴클레오티드에 상보적인 터미날 뉴클레오티드를 가지는 두번째 진단 프라이머 혹은 (b) 첫번째 핵산 서열의 진단 부위에 거의 상보적인 서열을 지니며, 상기 예견되는 변이 뉴클레오티드에 상응하는 정상적인 뉴클레오티드에 상보적인 터미날 뉴클레오티드를 가지는 첫번째 진단 프라이머 및 두번째 핵산 서열의 진단 부분에 거의 상보적인 서열을 지니며 상기 예견되는 변이 뉴클레오티드에 상응하는 정상적인 뉴클레오티드에 상보적인 터미날 뉴클레오티드를 가지는 두번째 진단 프라이머.
  4. 제1항에 있어서, 상기 예견되는 변이 뉴클레오티드를 포함하는 상기 샘플 핵산의 적어도 일부분을 먼저 증폭하고 수득한 증폭 생성물을 처리될 샘플로 사용하는 것을 포함하는 방법.
  5. 제3항에 있어서, 상기 예견되는 변이 뉴클레오티드를 포함하는 상기 동일 핵산이 적어도 일부분을 먼저 증폭하고 수득한 증폭 생성물을 처리될 샘플로서 사용하는 것을 포함하는 방법.
  6. 제1항에 있어서, 상기 샘플 핵산 처리를 적어도 다시한번 반복시켜서 상기 동일 표적 염기 서열을 주형으로 사용하여 확장 생성물을 선형 즉폭시키고 포함하는 방법.
  7. 제1항 내지 제6항중의 어느 한항에 있어서, 상기 예견되는 변이 뉴클레오티드 혹은 그 상응하는 정상적인 뉴클레오티드중 어느 하나에 상보적인 진단 프라이머의 터미날 뉴클레오티드가 상기 진단 프라이머의 3′ 말단에 존재하는 방법.
  8. 제6항에 있어서, 상기 샘플을 1,2 혹은 3가지 뉴클레오시드 트리포스페이트와 처리하여 형성되는 확장생성물이 상기 1,2 혹은 3가지 뉴클레오시드 트리포스페이트가 허용하는 범위에서만 확장될 수 있는 방법.
  9. 제8항에 있어서, 상기 샘플을 3가지 뉴클레오시드 트리포스페이트를 사용하여 처리하는 방법.
  10. 터미날 뉴클레오티드가 주지된 유전적 결함과 관련된 예견되는 변이 뉴클레오티드 혹은 그 상응하는 정상적인 뉴클레오티드중 어느 하나에 상보적이고, 상기 터미날 뉴클레오티드를 제외한 나머지 부분이 상기 예견되는 변이 뉴클레오티드 혹은 그 상응하는 정상적인 뉴클레오티드에 인접한 상기 나머지 부분과 상응하는 표적 염기 서열에 거의 상보적이고 사용될때는 본 발명의 방법에 있어서 진단 프라이머로서 존재하고, 상기 진단 프라이머의 상기 터미날 뉴클레오티드가 상기 표적 열기 서열의 그에 상응하는 뉴클레오티드에 상보적인 경우, 상기 진단 프라이머의 확장 생성물이 합성되지만, 상기 진단 프라이머의 상기 터미날 뉴클레오티드가 상기 표적 염기 서열의 그에 상응하는 뉴클레오티드와 상보적이지 않을 경우는 어떠한 확장 생성물도 합성되지 않는, 본 발명의 방법에서 사용되는 약5내지 50bp를 가지는 뉴클레오티드 서열.
  11. 제10항에 있어서, 상기 터미날 뉴클레오티드가 3′ 말단에 존재하는 뉴클레오티드 서열.
  12. 제10항 혹은 제11항에 있어서, 상기 예견되는 변이 뉴클레오티드가 정상적인 뉴클레오티드 서열의 점돌연변이(point mutation)로부터 유래한 뉴클레오티드 서열.
  13. 제10항 내지 제12항중 어느 한항에 있어서, 하나의 서열의 터미날 뉴클레오티드가 주지된 유전적 결함과 관련된, 예견되는 변이 뉴클레오티드에 상보적이고, 다른 하나의 서열의 터미날 뉴클레오티드가 상기 변이 뉴클레오티드에 상응하는 정상적인 뉴클레오티드에 상보적인 두가지 뉴클레오티드 서열의 세트.
  14. 샘플에 포함된 하나 혹은 그 이상의 핵산에서 적어도 하나의 변이 뉴클레오티드의 존재 유무를 검출하고, (1) 표적 염기 서열의 각 진단부분에 대한 진단 프라이머에 있어서, 각 진단 프라이머의 뉴클레오티드 서열이 상기 진단부분에 거의 상보적인 상태이고, 상기 진단 프라이머의 터미날 뉴클레오티드가 상기 예견되는 변이 뉴클레오티드 혹은 그에 상응하는 정상적인 뉴클레오티드중 어느 하나에 상보적인 상태이어서, 사용될 때, 진단 프라이머의 상기 터미날 뉴클레오티드가 표적 염기 서열의 사응하는 뉴클레오티드에 상보적인 경우는 진단 프라이머의 확장 생성물이 합성되지만, 상기 진단 프라이머의 상기 터미날 뉴클레오티드가 상기 표적 염기 서열의 상응하는 뉴클레오티드에 상보적이지 않은 경우는 어떠한 확장 생성물도 합성되지 않는, 상기 언급한 표적 염기 서열의 각 진단부분에 대한 진단 프라이머, (2) 4가지 상이한 뉴클레오시드 트리포스페이트, (3) 상기 뉴클레오시드 트리포스페이트를 중합화시키는 기능물을 포함하는 키트(kit).
  15. 제14항에 있어서, 하나의 진단 프라이머의 터미날 뉴클레오티드가 주지된 유전적 결합과 관련된 예견되는 변이 뉴클레오티드에 상보적이고, 다른 하나의 진단 프라이머의 터미날 뉴클레오티드가 상기 예견되는 변이 뉴클레오티드에 상응하는 정상적인 뉴클레오티드에 상보적인, 표적 염기 서열의 각 진단부분에 대한 상기 두가지 진단 프라이머 세트.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019890003001A 1988-03-10 1989-03-10 뉴클레오티드 서열을 검출하는 방법 KR970003151B1 (ko)

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