用改良消减杂交结合选择性PCR法构建老年性白内障消减cDNA文库

为构建含较多大片段的高质量的老年性白内障消减cDNA文库 ,利用生物素标记、磁珠分离的改良消减杂交法获得差异cDNA .利用选择性PCR法扩增其中大片段差异cDNA ,将其与T 载体进行T A连接并转化入大肠杆菌 ,成功构建老年性白内障消减cDNA文库 .共获得 4 0 0 0余个克隆 ,随机挑取的 2 2个克隆中 ,≥ 10 0 0bp的片段有 7个 ,占 31 8% ,≥ 75 0bp有 15个 ,占 6 8 2 % .将≥ 75 0bp的 15个克隆进行反向点杂交 ,排除其中假阳性克隆 ,阳性克隆经测序并与GenBank比较 ,得到 6个已知基因、1个新基因 ,6个已知基因中 4个为全长基因 ,说明所得cDNA片段较大 ,文库质量较高 .改良消减杂交法结合选择性PCR法可以快速有效地获得大片段高质量的消减cDNA文库 ,为进一步筛选、鉴定老年性白内障致病相关基因奠定了基础

Construction of a Subtractive cDNA Library of the Age-related Cataract by an Improved Subtractive Hybridization and Selective PCR

In order to obtain an efficient subtractive cDNA library with larger fragments of age related cataract, an improved subtractive hybridization with biotin labeling and magnetic bead isolation to obtain the subtractive cDNA was used. The large fragments of the subtractive cDNA were amplified by using selective PCR method and directly inserted into T/A cloning vector and transformed into E.coli . The library contained 4 000 clones. 22 clones were selected at random and identified by the restriction digestion, 7 fragments were ≥1 000 bp, being 31 8% of the total, and 15 fragments were ≥750 bp, being 68 2% of the total. The 15 clones with fragments ≥750 bp were hybridized by reverse dot blotting hybridization to remove the false positive fragments. The positive clones were sequenced and compared with GenBank. 6 cDNA fragments of known genes and a novel gene were obtained. 4 of the 6 known genes were full length gene. The cDNA fragments are larger and the library is efficient. A high qualitative subtractive cDNA library with larger fragments can be constructed rapidly and effectively by improved subtractive hybridization and selective PCR method. This lays a foundation for further screening and identifying the specific expressed gene of age related cataract.