[1]董志敏,李英慧 　 张宝石 　 关荣霞 　 常汝镇 　 邱丽娟.一种获得大片段克隆的 SMART 全长 cDNA文库构建方法*[J].大豆科学,2006,25(03):223-227.[doi:10.11861/j.issn.1000-9841.2006.03.0223]
　Dong Zhimin,Li Yinghui Zhang Baoshi Guan Rongxia Chang Ruzhen Qiu Lijuan.A IMPROVED SMART METHOD TO CONSTRUCTIONFULL-LENGTH cDNA LIBRARY FOR LARGE CLONES[J].Soybean Science,2006,25(03):223-227.[doi:10.11861/j.issn.1000-9841.2006.03.0223]
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一种获得大片段克隆的 SMART 全长 cDNA文库构建方法*

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第25卷 期数: 2006年03期 页码: 223-227 栏目: 出版日期: 2006-08-25
Title:
A IMPROVED SMART METHOD TO CONSTRUCTIONFULL-LENGTH cDNA LIBRARY FOR LARGE CLONES
作者:
董志敏 1;  2 　 李英慧 2 　 张宝石 1 　 关荣霞 2 　 常汝镇 2 　 邱丽娟 2
(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)
Author(s):
Dong Zhimin 1;  2 　 Li Yinghui 2 　 Zhang Baoshi 1 　 Guan Rongxia 2 　 Chang Ruzhen 2 　 Qiu Lijuan 2
(1. College of Agronomy , Shenyang Agricultural University , Shenyang 110161 ;2. National Key Facility of Crop Gene Resources and Genetic Improvement ,Key Laboratory of Crop Germplasm &Biotechnology (MOA)/ Institute of CropSciences, Chinese Academy of Agricultural Sciences , Beijing 100081)
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
Keywords:
Improved SMART ; Amplified cDNA size fractionation; Full-length cDNA library ; Largeclones
DOI:
10.11861/j.issn.1000-9841.2006.03.0223
文献标志码:
A
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 。
Abstract:
It is difficult to obtain large clones among the SMART library due to PCR amplification impai-ring the proportion of large clones. According to this, SMART method was improved based on the primarySMART method. The soybean leaves cDNAs were separated into three parts based on the size distributionof amplified dscDNA by agarose gel size fractionation. Each of three parts was ligated respectively to vec-tor and transformed to Ecoli. The sub-library contain about 1. 0 ×10 6 clones. The recombination ratio wasnearly 99%and full-length ratio was 53. 6 %which was correspondence with that of primary SMART li-brary. The inserts size ranged between 0. 5kb to 3. 5kb and the size of largest inserts was about 1. 5kb lar-ger than that of primary SMART method. The improved SMART method increased obviously the largeclone proportion among the library which was in favor of soybean fuctional genomics research.

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备注/Memo

基金项目:国家自然科学基金重大项目(30490250)和国家“863”计划项目(2003AA207060)

作者简介:董志敏(1978 -), 女, 在读博士, 主要从事大豆功能基因组研究。
通讯作者:邱丽娟, T el:010 - 62186650;E- mail:qiu_lijuan @263. net 。

更新日期/Last Update: 2016-12-15