一种改良的从LongSAGE标签中鉴定猪新基因的GLGI方法

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10 bp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 of gene expression (LongSAGE) and the gene ration of longer cDNA fragments from serial analysis of gene 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 pMD-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.