Institute of Bioengineering and Nanotechnology, The Nanos, Singapore, Singapore.
Anal Biochem. 2010 Nov 1;406(1):51-60. doi: 10.1016/j.ab.2010.06.036. Epub 2010 Jun 27.
Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6kbp from 1nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74kbp from 1nM oligonucleotide.
在这里，我们提出了一种简单、高效、通用的自动动力学开关（AKS）基因合成方法，该方法仅通过一个聚合酶链反应（PCR）过程，就能从 1nM 寡核苷酸合成长达 1.6kbp 的 DNA。该方法在一步法基因合成中消除了 PCR 组装和扩增之间的干扰，同时通过使用一对侧翼引物，最大限度地扩增出现的所需 DNA。此外，我们描述了一种基于 DNA 杂交热力学和动力学的 PCR 基因合成分析模型。使用该模型分析了标准 PCR 扩增和一步法 PCR 基因合成之间的动力学差异，并通过对八个基因片段（318-1656bp）的实时基因合成进行了验证。系统地研究了寡核苷酸浓度、退火温度的严格性、退火时间、延伸时间和 PCR 缓冲条件的影响。对实验结果的分析为基因合成过程提供了新的见解，并有助于优化基因合成条件。我们进一步将该方法扩展到多聚基因组装，从 1nM 寡核苷酸中合成总 DNA 长度可达 5.74kbp。
