直接获得差异表达基因全长cDNA的改良消减杂交方法

目的：探索一种可以直接克隆差异表达基因全长cDNA的方法。方法：结合Clontech公司的“SMART PCR”cDNA合成技术和Promega公司的生物素．磁珠亲和技术．以鼻咽癌耐药细胞系CNE2／DDP及其素代细胞CNE2为研究时象．采用一种基于PCR技术的改良消减杂交方法筛选并克隆耐药相关基因。点杂交鉴定排除假阳性．对部分差异表达片段测序并进行序列分析,RT-PCR对差异表达的基因片段做进一步验证。结果：用此方法建立了可以克隆差异表达基因cDNA全长的双向消减文库。经过对部分差异表达片段测序和序列分析．获得了6个耐药细胞差异表达的基因片断。其中5个有完整的开放读框．可能代表耐药相关的差异表达基因。对此5个序列登录GenBank并获得登录号为AY196929．196933。结论：改良的消减杂交方法是克隆差异表达基因的有效方法。应用此方法研究发现,有多个功能已知和未知的基因通过上调或降低其表达参与了鼻咽癌耐药性的产生。

A new strategy: PCR-based subtractive hybridization for obtaining full length differential cDNA directly

Objective: To investigate the possibility of a strategy for obtaining the full length differentially expressed cDNA directly. Methods: A new approach of improved subtractive hybridization, which combining the techniques of Clontech's "SMART PCR" cDNA synthesis and the Promega's biotin-magnetic beads, was employed to screen and clone human nasopharyngeal carcinoma drug-resistance genes on the multidrug resistance cell line CNE2/DDP and its parent CNE2. Sequencing and blast analysis were performed after the differentially expressed genes had been tested by dot blotting. The result was further confirmed by RT-PCR. Results: A double-way subtractive library was established, which could be used to clone the full length differentially expressed cDNA. After sequencing and blast analysis, 6 differentially expressed sequences were obtained, 5 of them were found to have complete open reading frame (ORF) and may represent the drug resistant relative genes. The 5 sequences were registered to the GenBank and numbered as AY196929-196933. Conclusion: The PCR-based improved subtractive hybridization is an effective approach to identify differentially expressed genes. With this strategy, it is discovered that many genes, function known and unknown, might contribute to the production of multidrug resistance phenotype of human nasopharyngeal carcinoma through increasing or decreasing their expressions.