Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment
Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment
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DOI：
10.1016/j.ab.2009.02.008
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发表时间：
2009-05-01
影响因子：
2.9
通讯作者：
Savilahti, Harri
中科院分区：
文献类型：
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作者：
Rasila, Tiina S.;Pajunen, Maria I.;Savilahti, Harri
随机突变方法构成了一个有价值的蛋白质修饰工具箱，其应用范围从蛋白质工程到定向蛋白质进化研究。虽然目前有多种技术，但该领域缺乏直接比较不同方法的性能参数和操作范围的研究。在这项研究中，我们仔细研究了几个最常用的随机诱变技术，严格评估流行的易错聚合酶链反应（PCR）协议，以及羟胺和一个突变大肠杆菌菌株诱变方法。使用允许直接比较方法的报告质粒分析相对突变频率。易错PCR方法产生最高的突变率和最宽的操作范围，而化学和生物方法产生的突变水平低，并表现出狭窄的操作范围。转换与颠换的库在方法之间变化，表明使用高多样性全面诱变的方法的组合。使用本研究中定义的参数，评价的诱变方法可用于受控诱变，其中诱导突变的预期平均频率可调整至理想水平。(C)2009 Elsevier Inc. All rights reserved.
Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are Currently available, the field is lacking Studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chemical and biological methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions versus transversions varied among the methods, Suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this Study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended average frequency of induced mutations can be adjusted to a desirable level. (C) 2009 Elsevier Inc. All rights reserved.