单向锚定 PCR 富集扩增在克隆低丰度基因转录本中的应用  

Cloning of Low Richness Transcripts by Using Uni lateral Anchored PCR Enrichment Amplification Method

作　　者：郑其平[1] 余龙[1] 姜春玲[1] 张宏来 毕安定[1] 赵勇[1] 庚镇城 赵寿元[1] 

机构地区：[1]复旦大学遗传工程国家重点实验室

出　　处：《高技术通讯》1998年第10期40-44,共5页Chinese High Technology Letters

基　　金：863计划资助项目;国家杰出青年基金项目;上海市科技发展重大项目

摘　　要：利用国际生物信息资源，通过计算机检索寻找与已知功能基因具有高度同源的ＥＳＴｓ，据此设计特异的ＰＣＲ扩增引物，并通过单侧特异引物搭配ｃＤＮＡ分子库载体引物（锚定引物），在低严紧条件下扩增各种组织的ｃＤＮＡ分子库以富集目标片段。继以单向锚定ＰＣＲ扩增产物为模板，用双侧特异引物再次扩增，获得的特异扩增ＰＣＲ产物经测序证实之后，将其用作探针杂交筛选相应的ｃＤＮＡ文库并进而克隆目的基因的全长ｃＤ－ＮＡ。此方法用于三个人类新基因的全长ｃＤＮＡ的分离与克隆，结果全部获得成功。这三个人类新基因分别是Ｈ－ＲａｌＧＤＳ基因、Ｈ－ＣＩＳ基因和Ｈ－Ｓ６ＰＫ基因，它们在ＧｅｎＢａｎｋ数据库的登录号分别是：ＡＦ０２７１６９、ＡＦ０３５９４６及ＡＦ０３７４４７。本文结果提示这一方法对于应用现有的人类基因组生物信息资源，克隆具有重要生物学功能的人类新基因，尤其是克隆呈低丰度的基因转录本全长序列具有较高的实用价值。Based on the bioinformatic resources, ESTs showed high homology to known important functional genes were searched by computer analysis, and specific PCR amplication primers were designed according to the DNA sequences of them. And then, low stringent PCR amplification method was used to amplify the different cDNA libraries by using the combined uni specific primer and anchored phage vector primers of the cDNA libraries, so as to enrich the target cDNA fragments. The uni anchored PCR products was used as a template for the followed stringent PCR amplication by using the specific primers. The specific cDNA fragments thus obtained and confirmed by the followed sequencing analysis could be used as hybridization probes to screen the corresponding cDNA library for cloning of full length cDNA of the target genes. By using the strategy metioned above, we have successfully cloned the full length cDNA of several human novel genes such as H RalGDS, H CIS and H S6PK gene, and they all have got accession numbers in GenBank which are AF027169, AF035946 and AF037447 respectively. The result of this paper indicated that, with the aid of present bioinformatic resources on human genome research, the strategy mentioned above would be of great value in cloning of novel human genes with important biological function, especially in the cloning of full length transcripts with low richness.

关 键 词：基因克隆 锚定PCR 低丰度转录本 扩增引物 

分 类 号：Q78[生物学—分子生物学]