乳液PCR扩增复杂基因序列的方法建立及普通PCR的比较

The Construction of the Emulsion PCR Amplification Complex Gene Sequence and Its Comparison with Conventional PCR

该研究分析普通PCR的不足,通过较多实验研究初步建立乳液PCR方法,改善PCR实验技术.采用人工合成的乳酸杆菌质粒为模板,不同成分配比条件下的乳液PCR和普通PCR对质粒序列进行扩增,对扩增产物进行琼脂糖凝胶电泳,电泳结束后对凝胶进行Gelred染色并用GS-800灰度扫描仪成像.进一步分析乳液PCR和普通PCR扩增产物的特异性以及不同成分配比条件下乳液PCR的扩增质量.通过比较两种PCR方法的扩增结果可得,在相同条件下,乳液PCR扩增特异性高于普通PCR扩增,并确定当水相中加入100 g/L BSA,水油体积比在1∶2.5时,破乳水饱和乙醚体积比为1∶1时,水油相在1700 rpm条件下连续混合5 min时扩增效果最好.

In the present study, the shortage of Amplification Complex Gene Sequence by Conventional PCR was analyzed, and then a protocol was constructed to minimize these problems. With the plasmid of Lactobacillus as a template, different components of the emulsion PCR and the conventional PCR plasmid template were effectively amplified to promote the amplified product with PCR to carry on agarose electrophoresis. With that accomplished, the Gelred dyeing was done to the gelatin and the image formation was obtained by a GS-800 gradation scanner. Then a further analysis was made to get the specialities of the emulsion PCR and the conventional PCR amplified products and the producing quality by emulsion PCR. In terms of different components, it was found out by contrasting the two methods that the speciality of the emulsion PCR amplification was higher than that of the conventional PCR with the same conditions. Moreover, when 100 g/1 BSA was added to aqueous phase with the volume ratio of water-in-oil（w/o） at 1：2.5, the volume ratio of breaking water-saturated diethyl ether at 1：1 and water and oil mixture was thoroughly mixed in 5 min at 1700 rpm, the best amplification effect was obtained.