一种用少量标本快速构建老年性白内障消减cDNA文库的方法  

A Rapid Procedure for The Construction of Subtractive cDNA Library of Age-related Cataract From Small Amounts of Sample

作　　者：刘燕[1] 周健[1] 刘新平[2] 药立波[2] 王吉村[2] 苏景宽[3] 惠延年[1] 

机构地区：[1]第四军医大学西京医院眼科,西安710032 [2]第四军医大学生物化学与分子生物学教研室,西安710032 [3]第四军医大学唐都医院医教部,西安710032

出　　处：《生物化学与生物物理进展》2002年第6期952-954,共3页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目 (3 0 0 0 0 185 )~~

摘　　要：为从少量标本中获得含较多大片段的、高质量的老年性白内障消减cDNA文库 ,利用磁珠分离、生物素标记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 ,从而成功构建老年性白内障消减cDNA文库 .在文库中随机挑取的 2 2个克隆中 ,10 0 0bp以上的片段有 7个 ,占 31 8% ,75 0bp以上有 15个 ,占 6 8 2 % .所得cDNA片段较大 ,可以满足下一步研究需要 .In order to obtain a quality subtractive cDNA library of age related cataract which contains more large fragments from small amounts of sample, an improved subtractive hybridization which using magnetic beads isolation and biotin labelling was used to obtain the subtractive cDNA. Then the large fragments of the subtractive cDNA were amplified by using selective PCR method, so the subtrative cDNA library of age related cataract was successfully constructed. 22 clones of the library were selected at random and were identified by the restriction enzymes digestion. 7 fragments were larger than 1 000 bp,which is 31 8% of the totals, 15 fragments were larger than 750 bp, which is 68 2% of the totals. The cDNA fragments are larger and can be used in further study. A subtractive cDNA library with high quality and more large fragments can be constructed rapidly and effectively by improved subtractive hybridization and selective PCR method from small amounts of sample.

关 键 词：少量标本 快速构建 老年性白内障 消减cDNA文库 改良消减杂交法 选择性PCR法 

分 类 号：R776.1[医药卫生—眼科] Q78[医药卫生—临床医学]