摘要
目的采用高效互补ＤＮＡ（ｃＤＮＡ）合成技术构建人前列腺癌的ｃＤＮＡ文库。方法从前列腺癌患者的经尿道电切标本中提取总ＲＮＡ，纯化后得到Ｐｏｌｙ（Ａ）＋ＲＮＡ；依次合成单链及双链ｃＤＮＡ；经纯化后与衔接头相连接；再与噬菌体载体λｇｔ１０臂相连接，于体外包装后建成ｃＤＮＡ文库；以大肠杆菌Ｃ６００ｈｆｌ－行滴度测试及扩增。结果构建的人前列腺癌ｃＤＮＡ文库含２７６×１０８重组子，克隆效率为３１９×１０１４重组子／ｇｃＤＮＡ。扩增后的文库浓度为４４×１０７重组子／μｌ，将文库稀释至１０－７时所产生的噬菌斑密度最为适宜。结论所构建的人前列腺癌高效ｃＤＮＡ文库为进一步筛选克隆前列腺癌的特异性癌基因或抗癌基因奠定了基础。
Objective To construct a cDNA library of human prostatic carcinoma with highly effective techniques of cDNA synthesis. Methods Total RNA of prostate carcinoma was extracted from the transurethral resection specimen of a patient and then poly (A) + RNA was extracted further.Moreover,single strand cDNAs and double strand cDNAs were synthesized in turn.After purified,double strand cDNAs were ligated to the specific linker,then with arms of the bacteriophage λgt 10 and further packaged in vitro to set up the cDNA library.Titering and amplification of the library was carried out with the E.Coli strain C600 hfl -. Results The titering showed that the library contatined 2.76×10 8 recombinants and the cloning efficiency was 3.19×10 14 recombinants/g cDNA.The amplified library was 4.4×10 7 recombinants /μl in concentration and the number of bacteriophage plagues was the most suitable in density after it was diluted to 10 -7 in concentration. Conclusions The constructed cDNA library of human prostate carcinoma is a highly efficient one and lays solid foundation for screening and cloning specific oncogenes or tumor suppressor genes of prostate carcinoma.
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
1999年第3期133-136,共4页
Chinese Journal of Urology
基金
国家自然科学基金
国家教委留学回国人员科研启动基金
关键词
前列腺肿瘤
CDNA文库
病因
Prostate neoplasms Carcinoma Complementary DNA Library
