Rong Shi, Wen-li Ma, Cui-hua Liu, Yan-bin Song, Xiang-ming Mao, Wen-ling Zheng
Institute of Molecular Biology, First Military Medical University, Guangzhou 510515, P.R. China.
J Biochem Mol Biol. 2004 Mar 31;37(2):148-52. doi: 10.5483/bmbrep.2004.37.2.148.
A new molecular-baiting method was studied by retrieving targeted gene fragments from an oligonucleotide microarray bait after hybridization. To make the microarray bait, 70-mer oligonucleotides that were designed to specifically represent the SSA1 gene of Saccharomyces cerevisiae were printed on the slide. Samples of the Saccharomyces cerevisiae mRNA were extracted and labeled by the RD-PCR (Restriction Display PCR) method using the Cy5-labelled universal primer, then applied for hybridization. The sample fragments that hybridized to the microarray were stripped, and the eluted cDNAs were retrieved and cloned into the pMD 18-T vector for transformation, plasmid preparation, and sequencing. BLAST searching of the GenBank database identified the retrieved fragments as being identical to the SSA1 gene (from 2057-2541bp). A new method is being established that can retrieve the sample fragments using an oligo-microarray-bait.
通过在杂交后从寡核苷酸微阵列诱饵中检索目标基因片段，研究了一种新的分子诱饵方法。为制作微阵列诱饵，将设计用于特异性代表酿酒酵母SSA1基因的70聚体寡核苷酸印在载玻片上。提取酿酒酵母mRNA样本，使用Cy5标记的通用引物通过RD-PCR（限制性显示PCR）方法进行标记，然后用于杂交。与微阵列杂交的样本片段被洗脱，回收洗脱的cDNA并克隆到pMD 18-T载体中进行转化、质粒制备和测序。对GenBank数据库进行BLAST搜索，确定回收的片段与SSA1基因（2057 - 2541bp）相同。正在建立一种可以使用寡核苷酸微阵列诱饵检索样本片段的新方法。
