摘要
目的快速构建成人胃癌组织cDNA文库。方法从人胃癌组织分离总RNA，以含有轴IB酶切位点的oligo（dT）引物合成第1链cDNA，以含蛳IA酶切位点的SMART寡核苷酸为引物经LD-PCR扩增双链cDNA，双链cDNA经sGI（IA＆IB）酶切，以CHROMA SPIN＋TE-1000柱分级分离cDNA，收集符合需要的cDNA片段并纯化，随后将之与λTriplEx2载体连接经体外包装成噬菌体λ文库。结果经检测共获得2．73×10^6个重组子，重组率约为94％，插入片段大小平均为1．2kb。结论用SMART方法构建的胃癌组织cDNA文库质量较高，为以后筛选胃癌相关基因奠定了基础。
Objective To quickly construct a directional eDNA library from human gastric carcinoma tissues. Methods The total RNA was separated from mixed human gastric carcinoma tissues, then the first strand cDNA was synthesized with oligo（dT） primer containing Sfi I-digested sites before the double-strand eDNA was amplified through LD-PCR （long-distance PCR） by SMART technology. The double-strand eDNA was digested by Sfi I（IA ＆IB） restriction enzyme before eDNA size fractionation, the double-strand eDNA fractionated was ligated into λTripIEx2 vector and packaged in vitro. Results The unamplified human gastric carcinoma tissues cDNA library consisted of 2. 73 x 10^6 independent clones with recombinant clones was 94%. The average size of the recombinants insert 1.2 kb. Conclusion The quality of the constructed human gastric carcinoma tissues cDNA library is excellent and helpful to screen human gastric carcinoma specific antigen.
出处
《中国临床新医学》
2011年第5期394-397,共4页
CHINESE JOURNAL OF NEW CLINICAL MEDICINE
基金
国家自然科学基金资助项目(编号:81060201)
广西科学研究与技术开发计划项目(编号:桂科攻10124001A-22)
中国博士后科学基金特别资助项目(编号:201003342)
关键词
CDNA文库
胃癌
定向克隆
cDNA library
Human gastric carcinoma
Directional cloning
