一种改良的从LongSAGE标签中鉴定猪新基因的GLGI方法(英文)  

A Modified GLGI Method for Identification of Novel Porcine Genes from Long Serial Analysis of Gene Expression Tags

作　　者：唐中林[1] 李勇[1] 赵书红[1] 刘榜[1] 樊斌[1] 李奎[1] 

机构地区：[1]华中农业大学动物遗传育种教育部重点实验室

出　　处：《农业生物技术学报》2007年第1期15-19,共5页Journal of Agricultural Biotechnology

基　　金：This work was supported by the Key Project of National Natural Science Foundation of China (No.30330440).

摘　　要：将长的基因表达序列标签(LongSAGE)和结合标签的基因序列延伸(GLGI)技术相结合,对GLGI的方法进行改良。(1)将原始GLGI方法中正向引物中长为10bp的SAGE标签特异序列改为17bp的LongSAGE标签;(2)针对不同LongSAGE标签,在PCR反应中给定相应不同的退火温度;(3)在PCR反应中用普通的DNA聚合酶代替高保真酶(Pfu);(4)在PCR反应的克隆中,使用普通的T载体和感受态细胞代替原始方法中特异的载体和感受态细胞。利用改良的GLGI方法分离的EST位于cDNA的3′末端并带有加尾信号和ploy(dA)尾巴。该方法在鉴定低丰度表达的基因时比普通的EST测序方法具有更高的灵敏性。Combining with the long serial analysis ofgene expression （LongSAGE） and the generation of longer cDNA fragments from serial analysis ofgene expression tags for gene identification （GLGI） technique, a new strategy called modified GLGI（M-GLGI） was developed to isolate unknown 3＇ EST and discover novel genes： （1） A 17-base pairs LongSAGE tag was used as sense primer instead of 10-base SAGE tag; （2） PCR reaction was performed under an appropriate annealing temperature for each tag; （3） Universal DNA polymerase was used in PCR amplification instead of Pfu enzyme; （4） Common cloning strategy using p^MD-18T vector and E.coli DH5α cell were used instead of a special vector and competent cells. Moreover, ESTs isolated by M-GLGI had 3＇ end with the polyadenylation signals and ploy （dA） tails. This method is more sensitive for identifying gene expressed in low abundance than conventional EST sequencing.

关 键 词：长的基因表达序列标签 GLGI改良 新基因鉴定 

分 类 号：S188[农业科学—农业基础科学]