Ye Hongye, Huang Mo Chao, Li Mo-Huang, Ying Jackie Y
Institute of Bioengineering and Nanotechnology, The Nanos, Singapore.
Nucleic Acids Res. 2009 Apr;37(7):e51. doi: 10.1093/nar/gkp118. Epub 2009 Mar 5.
Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10 degrees C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.
在此，我们展示了一种简单且经济高效的自上而下（TD）基因合成方法，该方法在一步法基因合成中消除了聚合酶链反应（PCR）组装与扩增之间的干扰。该方法包括两个关键步骤：（i）设计熔解温度差大于10摄氏度的外部引物和组装寡核苷酸组，以及（ii）利用退火温度来选择性地控制寡核苷酸组装和全长模板扩增的效率。此外，我们已将所提出的方法与实时PCR相结合，以分析基因合成过程的逐步效率和动力学。将凝胶电泳结果与实时荧光信号进行比较，以研究寡核苷酸浓度、外部引物浓度、退火温度的严格性以及PCR循环次数的影响。对实验结果的分析有助于深入了解基因合成过程。我们进一步讨论了防止形成假DNA产物的条件。TD实时基因合成方法为组装相当长的DNA序列提供了一种简单有效的方法，并有助于优化基因合成条件。据我们所知，这是首次利用实时PCR进行基因合成的报道。
