유전자 증폭 기술의 종류

목차

Ⅰ. PCR의 일반적인 thermal programming
Ⅱ. PCR기법의 응용
Ⅲ. References

본문내용

Ⅰ. PCR의 일반적인 thermal programming.
1. Hot Start
PCR은 동일한 parameter로 구성된 반복 cycle들을 통해서 이루어진다. 이것은 PCR machine의 thermal parameter가 반복된다는 의미이다. PCR reaction은 매 cycle마다 달라지게 된다. PCR의 components와 pH 등은 역동적으로 변화하면서 매번 새로운 환경으로 cycle을 지난다. 크게 이 역동적인 cycle들은 initial screening phase와 exponential replication phase 두 가지로 나누어 생각할 수 있다.
1) Initial screening phase에서는 primer가 target으로 하는 sequences를 찾아서 annealing하고 원하는 size의 target fragment를 만들어 내는 phase.
2) exponential replication phase는 만들어진 target fragment를 계속 복제해내는 phase.
이렇게 두가지로 크게 나누는 이유는 initial screening phase에서 PCR의 scnsitivity 혹은 yield와 specificity가 결정이 되기 때문이다. 이 phase에서 primer mismatch나 partial binding에 의해서 잘못된 fragments가 생겨나면 이후에서는 원하는 target fragment와 동일한 조건에서 같이 증폭이 되어 버리기 때문이다.

처음 annealing step에서 온도 값이 적절하다면 primer는 정확히 자기의 matching sequences에 binding합니다. 하지만 Tm값보다 낮은 온도에서 annealing을 시킬 경우 31 end의 부분적인 sequences만 matching 되더라도 primer의 partial binding이 일어난다.

참고자료

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