A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase
A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase
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DOI：
10.1042/ba20040195
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发表时间：
2005-12-01
影响因子：
2.8
通讯作者：
Li, MG
中科院分区：
文献类型：
--
作者：
Yang, SH;Li, X;Li, MG
本文报道了一种从双链DNA的粘性末端制备平末端的高效方法。Klenow片段和Pfu DNA聚合酶用于填充粘性末端。由于转化效率可以直接反映填充效率，因此使用类似的连接和转化条件，并将填充效率与相应的转化效率进行比较。结果表明，PfuDNA聚合酶的填充效率是Klenow片段的1.96倍，明显高于Klenow片段（P < 0.01）。对反应条件的优化实验表明，当pH为8.5，温度为WC时，使用含有3mM MgSO_4和300 μ M dNTP的缓冲液时，填充效率最高。
The present paper reports a highly efficient method of making blunt ends from cohesive ends of double-stranded DNA. Klenow fragment and Pfu DNA polymerases were used to fill in the cohesive ends. Since the transformation efficiency can directly reflect the filling-in efficiency, similar ligation and transformation conditions were used, and the filling-in efficiency was compared with the corresponding transformation efficiency. The results indicate that the filling-in efficiency of Pfu DNA polymerase was 1.96 times that of Klenow fragment and its efficiency was markedly higher than that of Klenow fragment (P < 0.01). The optimization experiments on reaction conditions indicate, when the pH is 8.5 and the temperature is WC, that the filling-in efficiency was highest upon using a buffer containing 3 mM MgSO4 and 300 mu M dNTP.