产物变性胶纯化实现连续PCR的多轮扩增

More Round of Re-Amplificaiton for Consecutive PCR by Denatured-gel Purification

产物弥散成片是PCR操作中一种比较常见的异常现象,在连续PCR实验中会导致扩增的最终失败。这种异常产生的内在机制是扩增过程中非全长部分链的合成、积累及其对后续扩增的干扰。缓解这种PCR扩增异常现象的途径包括抑制非全长链的合成,以及在重复扩增之前去除模板中的非全长链成分。本文显示通过碱变性琼脂糖电泳然后割胶纯化获得下一轮扩增模板的方式能够有效地避免连续扩增中产物弥散成片的异常扩增。为有效地进行重复PCR扩增提供了一个新的途径。结果进一步证实了正常PCR扩增中非全长链产生的不可避免及其对新生链合成的干扰效应。

Smearing product is often obtained for PCR. Consecutive PCR would abort if smearing product is used as template for next rounds of amplification. Smearing product result from the synthesis of partial strar, ds during the process of polymerization of nascent strands. The ways to alleviate this abnormal phenomenon include to depress the synthesis of partial strands during thermal cycles within PCR, and to exlude those partial strands before it is used as template for next round of amplification. This paper showed that elimination of partial strands from product by elctrophoresis with alkali denatured agarose gel was effective, and then consecutive PCR could be carried out more rounds. Our results provide one new method to perform an effective re-amplification by PCR. Furthermore, these results also verify that the synthesis of partial strands is not avoided and it could distrub the synthesis of nascent strands.