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利用分隔式自我复制(CSR)改造来自于Thermus thermophilus的I型DNA聚合酶

在基因元件(例如DNA聚合酶)的定向进化中,CSR(分隔式自我复制)和CPR(分隔式同伴复制)是一类重要的方法。目前利用野生型的Taq DNA聚合酶进行乳化PCR仍存在运行时间长,PCR产物产量低且由于抑制剂的干扰存在假阴性的等缺陷。

该篇论文改进了来自于Thermus thermophilus HB27的I型DNA聚合酶 Tth pol,使其能更高效地用于DNA聚合酶与其他基因元件的定向进化。

图一 CSR定向进化流程图

在这一工作中,研究者首先进行了以下4个步骤的改进和突变(如图二):

i.删除N端5’-3’外切酶结构域;

ii.与DNA结合蛋白(Sso7d)融合表达;

iii.在Tth pol引入已知的能提高Taq pol活性的位点(626E, 707I, 708E, 743A);

iv.优化密码子降低模板GC含量 (从68%降低至53%)

图二 N端5’-3’外切酶结构域,Sso7d融合表达,以及引入已知的能提高Taq pol活性的位点;

经过以上的改进和突变,Tth pol的扩增效率得到了显著提高,如图三所示:

 

图三 改进前Tthpol和改进后 Tth pol的扩增能力对比

然后,研究者又构建了随机突变文库并利用CSR进行进一步的筛选,以期望获得更高效率的Tth pol突变体,能实现利用更少的循环数取得良好的扩增效果,且表现更稳定。

图四 CSR筛选SDTthCs12RsEx pol的随机突变文库

经过4轮筛选,在DNA结合蛋白Sso7d上的K7T和S18T,以及在Tth DNA聚合酶上的E338K和E690D,四个点突变被筛选到,表明这些突变体能在更短的循环数内达到较好的扩增效果。

图五 筛选到的突变体的突变位点(预测)

然后,研究者通过定点突变构建了单独的突变体,并进行了乳化和非乳化PCR的验证。

图六 非乳化和乳化PCR的验证

研究者发现,无论是乳化和非乳化PCR,Tth pol的突变体E338K和E690D都能够以10s/kb的速度扩增长度为4.65 kb DNA,表明这两个突变体能在更短的循环数内达到较好的扩增效果,然而其机制尚不清楚。

作者: 李新佳



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