摘要
目的 采用SMART (switchingmechanismat5′endofRNAtranscript)技术 ,快速构建了高质量的成人鼻咽上皮组织定向cDNA文库。方法 从成人正常鼻咽上皮组织分离总RNA ,利用经修饰的oligo(dT)引物 (含sfiIB酶切位点 )合成cDNA第一链 ,同时根据真核生物mRNA 5′端帽子结构特点 ,利用SMART核苷酸 (含sfiIA酶切位点 )作为cDNA第二链在mRNA 5′端延伸出去的模板 ,进而以此序列为引物利用LD PCR(Long distance PCR)合成双链cDNA ,双链cDNA经sfiI(IA&IB)酶切和过柱分级分离后 ,克隆入经sfiI酶切的λTripIEX2载体后经体外包装而成cDNA文库。结果 获得 1.5× 10 6个重组子 ,重组率达 10 0 %。文库扩增后 ,滴度达 3.8× 10 9pfu/ml,插入cDNA平均长度为 1.5kb。结论构建的成人鼻咽cDNA文库具有良好的质量 ,该cDNA文库为进一步筛选。
Objective To construct a directional cDNA library from human adult nasopharynx by SMART (switching mechanism at 5′ end of RNA transcript) technique. Methods The total RNA was separated from human adult nasopharynx epithelial fissue and the frist strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer(contained sfi IB site) while the SMART oligonudeotide(contained sfi IA site) was utilized as a template so that the frist strand cDNA could be extended over the 5′end of mRNA. The double strand cDNA was amplified by LD PCR(long distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction enzyme.After cDNA size fractionation through CHROMA SPIN column,the double strand cDNA was ligated into the sfi I digested λTripIEx2 vector and then the recombinant DNA was packaged in vitro. Results The unamplified human adult nasopharynx cDNA library consists of 1.5×10 6 independent clones in which the percentage of recombinant clones is about 100%.The titer of the amplified cDNA library is 3.8×10 9 pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. Conclusion These results shows that the human adult nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma(NPC) and tissue specific genes of human nasopharynx.
出处
《中华耳鼻咽喉科杂志》
CSCD
北大核心
2001年第1期47-50,共4页
Chinese Journal of Otorhinolaryngology
基金
国家863项目(102-10-01-05)
973重点项目"疾病基因组学"理论和技术体系的建立资助项目
