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目的采用SMART(switching mechanism at 5’ end of RNA transcript)技术,快速构建高质量的人脑胶质瘤组织全长cDNA文库。方法提取多份人脑胶质瘤组织标本的总RNA,混合后处理得到纯化的mRNA,利用CDSⅢ/3’PCR引物(含SfiⅠB酶切位点)反转录,用SMARTⅣOligo(dT)(含SfiⅠA酶切位点)作为mRNA5’端延伸出去的模板,合成多出一段SMARTⅣOligo(dT)互补序列的cDNA第一链,进而以此序列为引物合成全长的双链cDNA。双链cDNA经SfiⅠ(A&B)酶切后克隆入经SfiⅠ酶切的λTriplEx2载体,再经噬菌体包装蛋白包装成为cDNA文库。结果获得2.4×106个重组子,重组率达100%,文库扩增后,滴度达4.5×109 pfu/ml,插入cDNA平均长度为1.2kb。结论成功构建高质量的人脑胶质瘤组织全长cDNA文库,为进一步筛选、克隆人脑胶质瘤抑癌基因及特异性表达基因奠定基础。
Objective To rapidly construct a high-quality full-length cDNA library of human glioma tissues by using SMART (switching mechanism at 5 ’end of RNA transcript) technology. Methods Total RNA was extracted from multiple human glioma tissue samples and mixed and processed to obtain purified mRNA. Reverse transcription using CDSⅢ / 3’PCR primer (containing SfiⅠB restriction enzyme site) was performed. SMARTⅣOligo (dT) containing SfiⅠA Cleavage site) as a template for the extension of the mRNA 5’-end to synthesize the first strand of the cDNA which is complementary to the SMART IV Oligo (dT) sequence and synthesize the full-length double-stranded cDNA using the sequence as a primer. The double-stranded cDNA was digested with SfiI (A & B), cloned into the Srip-digested λTriplEx2 vector, and packaged into a cDNA library by phage packaging protein. Results 2.4 × 106 recombinants were obtained and the recombination rate was 100%. After amplification, the titer reached 4.5 × 109 pfu / ml, and the average length of inserted cDNA was 1.2kb. Conclusion The successful construction of a full-length cDNA library of human glioma tissue will lay the foundation for the further screening and cloning of human glioma tumor suppressor genes and specifically expressed genes.