Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine
Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine
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DOI：
10.1038/nmeth.4015
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发表时间：
2016-11-01
期刊：
影响因子：
48
通讯作者：
Gurnett, Christina A.
中科院分区：
文献类型：
--
作者：
Haller, Gabe;Alvarado, David;Gurnett, Christina A.
目标DNA序列的大规模突变使研究人员能够全面评估单核苷酸变化的影响。在这里，我们展示了使用可逆终止的脱氧肌苷三磷酸(RtITP)的大规模平行单核苷酸突变构建系统等位基因序列(SAS)。我们创建了一个突变文库，其中包含围绕TEM1β-内酰胺酶基因活性部位的每一个可能的单核苷酸突变。当与高通量功能分析相结合时，SAS突变文库可以加快遗传变异的功能评估。
Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 beta-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.