应用70 mer Oligo基因芯片从限制性cDNA片段中钓取目的基因

目的 探讨采用单一探针寡核苷酸阵列从经限制性显示方法制备的cDNA片段中钓取目的基因片段的方法。方法酵母经常规培养,抽提mRNA逆转录成cDNA后,经限制性显示技术制备成限制性cDNA片段,根据目的基因SSA1设计70mer特异性oligo片段并打印成为寡核苷酸阵列。采用荧光标记的通用引物对上述经限制性显示技术获得的限制性cDNA片段进行标记,与oligo阵列杂交,洗脱,扫描。然后进行杂交后剥除,收集剥除液,采用限制性通用引物扩增,产物克隆于PUC18T载体中,并转化至JM109大肠杆菌中培养,抽提质粒,进行测序鉴定。结果 测序结果BLAST同源性比较表明,用该方法成功的克隆出了目的基因片段。结论 采用70mer寡核苷酸芯片可以直接从限制性显示方法处理的cDNA片段中钓取目的基因,而无需构建cDNA文库。另外,本方法也可用于oligo芯片的杂交后对有表达差异的基因的获取及研究中。

Isolation of the target gene from cDNA restriction fragments using 70-mer oligo microarray

Objective To study the method for using a 70-mer oligo microarray as the probe to isolate target genes from the cDNA restriction fragments. Method Samples of Saccharomyces cerevisiae mRNA was extracted after heat shock culture and reversely transcribed into the double-stranded cDNAs, which were prepared into restriction cDNA fragments using restriction display (RD) method. The microarray was printed using a single 70-mer specific oligo designed to according to the SSA1 gene of yeast. The cDNA restriction fragments were labeled by PCR method with the Cy5 universal primer before hybridization with the microarray. The microarray was stripped after washing and scanning, and the strip solution was collected for another round of PCR amplification using the universal primer without fluorescence. The PCR product was then cloned into PUC18 T vector and transformed into to E.coli JM109 cells for amplification, and the plasmids were extracted and sequenced for identification. Results BLAST results showed that the target gene was cloned successfully. Conclusion The target gene can be isolated directly using the 70-mer oligo microarray as the probe from the cDNA fragments prepared by RD method, without the necessity of building a cDNA library. This method can also be used in further research to acquire the differentially expressed genes after the oligo microarray hybridization.