应用复合增强剂扩增人巨细胞病毒pp65全基因

在PCR过程中 ,模板GC含量过高是一个不利因素。如果设计扩增片段较长 ,则进一步增加了PCR扩增的难度。解决这一问题对于以PCR成功获取富含GC的长基因有非常重要的意义。以人巨细胞病毒pp65全基因 (约 1 95 0bp ,GC %为 67%)为例 ,在PCR系统中测试不同添加剂(甘油、乙醇、DMSO、甜菜碱等 )及各种组合 ,摸索扩增目的基因的最佳条件。结果发现 :无或单一的添加剂都不能获得目的基因片段 ,只有当同时使用DMSO和甜菜碱 ,并在适当浓度时才能够获得特异产物。在PCR系统中包含复合增强剂能有助于高GC %、长基因片段的扩增 ,为解决此类问题提供了一种有效的途径。

Combine Different Enhancers in PCR Reaction to Amplify HCMV pp65 Gene

High GC content in gene segments usually poses a adverse effect on PCR amplification, especially when the desired product is long. It is very useful to provide a effective method to overcome this difficulty. To amplify the whole pp65 gene (with GC% about 67%, 1950bp) of HCMV, various kinds of additives and the combination of them were included in the PCR reaction mixture to improve the PCR result. The specific PCR products couldn't be obtained if there was no additives or only one kind of additive used alone, while combining the betaine and DMSO together with optimal concentration, the specific amplicon with expected size was acquired. Therefore, proper additives in PCR system can overcome difficulties caused by high GC% template, and achieve long target gene.;