Randomization of genes by PCR mutagenesis.
Randomization of genes by PCR mutagenesis.
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DOI：
10.1101/gr.2.1.28
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发表时间：
1992-08-01
期刊：
影响因子：
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通讯作者：
Joyce, G F
中科院分区：
文献类型：
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作者：
Cadwell, R C;Joyce, G F
建立了一种将随机点突变引入克隆基因的改良聚合酶链式反应(PCR)方法。进行这些修改是为了在DNA合成过程中降低Taq聚合酶的保真度，而不会显著降低在PCR中实现的扩增水平。由此产生的聚合酶链式反应产物可以被克隆以产生随机突变文库，或者如果T7启动子被掺入适当的聚合酶链式反应引物中，则可以直接转录。用该方法对四膜虫核酶基因进行了突变，突变率为0.66%+/-0.13%(95%C.I.)通过序列分析确定的每个位置的每个聚合酶链式反应。对于碱基取代的类型，没有强烈的偏好。每个DNA序列的突变数量服从泊松分布，并且突变随机分布在扩增序列中。
A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes. The modifications were made to decrease the fidelity of Taq polymerase during DNA synthesis without significantly decreasing the level of amplification achieved in the PCR. The resulting PCR products can be cloned to produce random mutant libraries or transcribed directly if a T7 promoter is incorporated within the appropriate PCR primer. We used this method to mutagenize the gene that encodes the Tetrahymena ribozyme with a mutation rate of 0.66% +/- 0.13% (95% C.I.) per position per PCR, as determined by sequence analysis. There are no strong preferneces with respect to the type of base substituion. The number of mutations per DNA sequence follows a Poisson distribution and the mutations are randomly distributed throughout the amplified sequence.