巢式PCR

巢式PCR

Definition of Nested PCR :巢式PCR的定义:

Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (i nstead of one pair) of PCR primers are used to amplify a fragment.

The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers (as they lie / are nested withi n the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.

The advantage of nested PCR is that if the wrong PCR fragment was amplified, the pr obability is quite low that the region would be amplified a second time by the second set of primers. Thus, Nested PCR is a very specific PCR amplification.

The Nested PCR Reaction

Nested PCR requires two sets of primers which are used to amplify a specific DNA fr agment using two

separate runs of PCR.,. The second pair of primers function to amplify a smaller speci fic DNA fragment located within the first PCR product.

Nested PCR Reaction Diagram巢式聚合酶链反应图

Steps of the Nested PCR巢式PCR的步骤

Step One: The DNA target template is bound by the first set of primers shown in blue .. The primers may bind to alternative, similar primer binding sites which give multipl e products however only one of these PCR products give the intended sequence ( multiple products not shown).

Step Two： PCR products from the first PCR reaction are subjected to a second PCR run however with a second new set of primers shown in red

As these primers are NESTED within the first PCR product, they make it very unlikel y that non-specifically amplified PCR product would contain binding s ites for both sets of primers.. This nested PCR amplification ensures that the PCR pro duct from the second PCR amplification has little or no contamination from non-spe cifically amplified PCR products from alternative primer target sequences.

巢式PCR是一种变异的聚合酶链反应(PCR),这两组(而非一对)PCR引物可用来扩增片段.第一对PCR引物扩增的片段与片段准相近. 第二对引称为巢式引物(因为他们巢嵌在第一片段内部). 与第一段PCR产物结合，来指导第二段PCR产物的合成，这段产物比第一段产物要短。巢式PCR的好处是,如果错误的PCR产物被扩增，那么由第二段引物指导的第二次扩增的产物错误的可能性将是非常低的.巢式聚合酶链反应巢式PCR需要两套引物用于放大特定的DNA 片，应用两次单独的PCR来进行。第二对引功能扩增较小的特定DNA片段，该片段位于一次PCR产物内部因此,巢式PCR扩增是非常特异的.第一步:目标DNA片段与第一段引物（兰色）结合。引物可能回选择性的结合于模板，相似的引物结合位点会产生多重的产物，然而，只有一段产物是我们预期的。第二步：应用一次PCR产物区段，通过二次新的引物结合位点进行二次PCR（红色）。这些引物嵌套在一次PCR产物的内部，因此，非特异性片段的产生是不太可能的，一次PCR产物包含着二次PCR引物的结合位点。这种巢式PCR产物保证了通过二次PCR得到的产物很少有或者没有污染（杂带，也就是产物是符合我们需要的）也就是没有非特异性的条带