提高由芯片合成的寡核苷酸及其组装而成的基因DNA的保真性

项目名称： 提高由芯片合成的寡核苷酸及其组装而成的基因DNA的保真性

项目编号： No.31270149

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 洪泂

作者单位： 中国科学技术大学

项目金额： 80万元

中文摘要： 随着合成生物学等学科的发展，几十Kb，几百Kb长度的DNA的化学合成需求越来越大，但是基因DNA合成的高成本限制了此技术大范围的应用。在基因DNA的合成中，错误的测定和纠正是费用高昂的主要原因之一。通过芯片合成寡核苷酸可以大规模降低寡核苷酸合成成本，但难以降低错误的测定和纠正的费用。本课题针对此问题，采用错配结合蛋白MutS结合并去除有错寡核苷酸及DNA短片段的策略来大幅提高基因合成的保真性。通过克隆两种不同的错配结合蛋白MutS的基因，与纤维素结合域及绿色荧光蛋白融合表达，利用常规方法合成的寡核苷酸来验证MutS融合蛋白的功能，再建立优化的错配DNA结合条件及MutS组合，以去除芯片合成的有错寡核苷酸，并使用错误率大幅降低的寡核苷酸进行基因DNA合成。本项目预期建立一个简单快速的去除有错寡核苷酸和DNA片段的方法，为利用芯片合成的寡核苷酸低成本快速大规模高保真合成基因DNA打下基础。

中文关键词： DNA 合成；芯片；高通量；；

英文摘要： The development of economical and high-throughput de novo gene synthesis has been limited by the high errors in the synthesized DNA, and the error correction is a time and labor cost procedure. Oligonucleotide synthesis on Chip can reduce the cost for oligonucleotide greatly, but the low quality of synthesized oligonucleotide needs more error correction. In this project, two kinds of mismatch DNA binding proteins MutS are used in a method to correct the error in Chip synthesized oligonucleotides. The MutS are fused with CBM3 and EGFP to improve the expression and purification. The oligonucleotides synthesized by traditional method are used to construct the DS-DNA with various mismatchs for validation of the MutS function. And also these DS-DNA are also used to optimize the two kinds of MutS composition and the procedure of error correction. Then the constructed procedure is used in the error correction of the oligonucleotides for gene assemble, and high throughput error correction method will also been evaluated in the oligonucleotides for more and longer genes. Further more, the error correction for DNA fragment will be constructed. In this project, a high throughput error correction will be constructed for oligonucleotides and DNA fragment. It will be an important fundament for high throughput DNA synthesis.

英文关键词： DNA de novo synthesis；mircochip；high throughput；；