A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step.
A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step.
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DOI：
10.1186/1472-6750-4-2
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发表时间：
2004-02-26
影响因子：
3.5
通讯作者：
Duggleby RG
中科院分区：
文献类型：
--
作者：
Tyagi R;Lai R;Duggleby RG
定点诱变是改变基因结构和功能的有效方法。在这里，我们报告了一种快速高效的基于大引物的聚合酶链式反应 (PCR) 诱变策略，该策略绕过了两轮 PCR 之间任何 DNA 中间纯化。该策略依赖于使用限制浓度的侧翼引物（反向或正向）以及诱变引物的正常浓度，加上第一个 PCR 扩增步骤中延长的最终延伸周期。第一轮 PCR 生成大引物，随后与第二个侧翼引物一起用于第二轮 PCR，但无需对大引物进行中间纯化。该策略已成功用于四种不同的质粒，以产生各种突变体。该策略提供了一种非常快速、廉价且有效的方法来进行定点诱变。该策略提供了传统基于大引物的定点诱变的替代方案，该诱变基于中间凝胶纯化步骤。该策略产生高频率的诱变。
Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR. The strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants. This strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.
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影响因子：
14.9
作者：
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通讯作者：
KandelsLewis, S
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作者：
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