Cherry Jessica, Nieuwenhuijsen Bart W, Kaftan Edward J, Kennedy Jeffrey D, Chanda Pranab K
Neuroscience Discovery Research Wyeth Research, CN8000, Princeton, NJ 08543, USA.
J Biochem Biophys Methods. 2008 Apr 24;70(6):820-2. doi: 10.1016/j.jprot.2007.12.009. Epub 2008 Jan 9.
Here we report an improved, reproducible, simple, rapid, and cost-effective PCR-based DNA synthesis method using short (25-40 bp) overlapping oligodeoxyribonucleotides (oligos). The method involves two steps; (1) assembly of multiple/overlapping oligos by PCR to generate the template DNA and (2) amplification of the template DNA sequence with the two outermost oligos as primers. We have tested this method by synthesizing approximately 35 genes ranging in size between 300 bp and 1700 bp and G+C content from moderate (30%) to high (65%). In addition, we used the method to introduce 29 mutations simultaneously into a single gene. Key to the success of this method is the use of optimized oligo concentrations and the type of DNA polymerase used. This simplified and highly reproducible method is expected to be beneficial for the synthesis of a wide variety of genes.
在此，我们报告一种改进的、可重复的、简单、快速且经济高效的基于聚合酶链式反应（PCR）的DNA合成方法，该方法使用短（25 - 40个碱基对）的重叠寡脱氧核糖核苷酸（寡核苷酸）。该方法包括两个步骤：（1）通过PCR组装多个/重叠寡核苷酸以生成模板DNA；（2）以两个最外侧的寡核苷酸作为引物扩增模板DNA序列。我们通过合成大约35个基因对该方法进行了测试，这些基因的大小在300碱基对至1700碱基对之间，鸟嘌呤与胞嘧啶（G + C）含量从中等（30%）到高（65%）。此外，我们使用该方法同时将29个突变引入单个基因。该方法成功的关键在于使用优化的寡核苷酸浓度和所用DNA聚合酶的类型。这种简化且高度可重复的方法有望对多种基因的合成有益。
