一种获得大片段克隆的SMART全长cDNA文库构建方法  

A IMPROVED SMART METHOD TO CONSTRUCTION FULL-LENGTH cDNA LIBRARY FOR LARGE CLONES

作　　者：董志敏[1] 李英慧[2] 张宝石[1] 关荣霞[2] 常汝镇[2] 邱丽娟[2] 

机构地区：[1]沈阳农业大学农学院,沈阳110161 [2]中国农业科学院作物科学研究所国家农作物基因资源与遗传改良重大科学工程农业部作物种质资源与生物技术重点开放实验室,北京100081

出　　处：《大豆科学》2006年第3期223-227,共5页Soybean Science

基　　金：国家自然科学基金重大项目(30490250);国家"863"计划项目(2003AA207060)

摘　　要：针对SMART方法中PCR扩增削减了大片段基因所占比例，使文库中很难获得大片段克隆的问题，进行了方法改进。在原始SMART方法的基础上，以大豆叶片为材料，通过琼脂糖凝胶分级分离技术，将PCR扩增后合成的dscDNA分成3个等级，分别与载体连接、转化，构建相应亚库，每个亚库容量在1．0×10^6左右，重组率接近99％。改进方法所建文库的平均全长率53．6％，与改进前的（52．2%）相当。插入片段范围为0．5～3．5kb，最大插入片断长度比改进前的提高1．5kb。该方法提高了SMART方法中大片段克隆的获得率，为大豆功能基因组学研究提供了保障。It is difficult to obtain large clones among the SMART library due to PCR amplification impairing the proportion of large clones. According to this, SMART method was improved based on the primary SMART method. The soybean leaves cDNAs were separated into three parts based on the size distribution of amplified dscDNA by agarose gel size fractionation. Each of three parts was ligated respectively to vector and transformed to Ecoli. The sub-library contain about 1.0×10^6 clones. The recombination ratio was nearly 99% and full-length ratio was 53.6% which was correspondence with that of primary SMART library. The inserts size ranged between 0.5kb to 3.5kb and the size of largest inserts was about 1.5kb larger than that of primary SMART method. The clone proportion among tile library which was in improved SMART method increased obviously the large favor of soybean fuctional genomics research.

关 键 词：改进的SMART法 扩增后cDNA分级分离 全长CDNA文库 大片段克隆 

分 类 号：S565.1[农业科学—作物学]