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PCR原理及反應步驟

更新時(shí)間:2022-07-21      點(diǎn)擊次數:2801
  PCR的原理:
 
  多聚酶鏈式反應(polymerase chain reaction)簡(jiǎn)稱(chēng)PCR技術(shù),是近30多年發(fā)展起來(lái)的一種體外擴增特異性DNA片段的技術(shù),可在數小時(shí)之內對僅有幾個(gè)拷貝的基因放大千萬(wàn)倍。那么,PCR的原理是怎樣的呢?
 
  PCR技術(shù)實(shí)際上是在模板DNA、引物以及原料(四種脫氧核糖核苷酸)在適宜的反應條件下,通過(guò)DNA聚合酶的酶促反應完成DNA擴增的過(guò)程。
 
  PCR技術(shù)的特異性取決于引物和模板DNA結合的特異性。PCR反應分為三步:
 
  1)變性:通過(guò)加熱時(shí)DNA雙螺旋的氫鍵斷裂,雙鏈解離成單鏈DNA;
 
  2)退火:當溫度突然降低時(shí)由于模板分子結構較引物復雜的多,而且反應體系中引物DNA量遠大于模板DNA,使引物和其互補的模板在局部形成雜交鏈,而模板DNA雙鏈之間互補的機會(huì )較少;
 
  3)延伸:在DNA聚合酶和四種脫氧核糖核苷酸第五以及Mg2+存在的條件下,5’→3’的聚合酶催化以引物為起點(diǎn)的DNA鏈延伸反應。
 
  4)3步為一個(gè)循環(huán),每一個(gè)循環(huán)的產(chǎn)物可以作為下一個(gè)循環(huán)的模板,30個(gè)循環(huán)左右,介于兩個(gè)引物之間的特異性DNA片段得到了大量的復制,數量可達2×107個(gè)拷貝。
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