2种PCR方法扩增盐藻肌动蛋白基因3’旁侧序列比较  

The comparison between two techniques in isolating 3' flanking region of Dunaliella salina actin gene

作　　者：姜国忠[1] 谢华[1] 郭玉忠[1] 范天黎[1] 薛乐勋[1] 

机构地区：[1]郑州大学细胞生物学研究室,郑州450052

出　　处：《郑州大学学报（医学版）》2004年第1期41-44,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：河南省重大科技攻关基金资助项目 0 12 2 0 3 2 5 0 0 ;河南省杰出人才创新基金资助项目 0 2 2 10 0 190 0

摘　　要：目的 :比较扩增盐藻肌动蛋白基因 3’旁侧序列的 2种PCR方法。方法 :用pvuⅡ、EcoRⅤ和StuⅠ 3种限制性内切酶消化盐藻基因组DNA后 ,供 2种PCR用 ,一种是连接介导法PCR(LMPCR) ,与用人工合成的适配子相连并用适配子引物和盐藻肌动蛋白基因特异引物扩增未知序列 ;另一种是反向PCR(IPCR) ,酶切后的DNA自身环化做模板 ,用 2对基因特异引物反向扩增。结果 :2轮PCR后 ,LMPCR中得到大量的非特异扩增产物 ,测序结果发现许多产物是由适配子引物AP2单独扩增引起。而反向PCR在得到pvuⅡ和StuⅠ消化的自连文库中扩增得到 2 .5kb特异产物 ,经测序发现 ,片段两侧为基因特异引物 ,部分序列与已知序列相一致。Southern也进一步证实了IPCR扩增片段来源于盐藻基因组DNA。结论 :IPCR技术在克隆基因旁侧序列时优于LMPCR方法。Aim: The present study was to compare two PCR techniques in isolating the unknown sequences of 3' flanking region of Dunaliella salina (D.salina) actin gene. Methods: ligation-mediated PCR (LMPCR), with adaptors and gene-specific primers (GSP), and inverse PCR (IPCR) with two pairs of GSPs were performed using D.salina genome DNA as the templates which digested with restriction enzymes pvuII, EcoRV and StuI, followed by ligating with adaptors and circling themselves, respectively. Results: Using of LMPCR produced lots of nonspecific fragments after two rounds of PCR. Furthermore, most of them were amplified by single AP2 primer according to DNA sequencing. In contrast, 2.5 kb of specific fragments were obtained by IPCR from self-ligated libraries digested with pvuII and StuI, respectively, and sequencing results confirmed the two gene specific primers and its known sequence. In addition, Southern bloting revealed that the product by IPCR was derived from D.salina genome DNA. Conclusion: IPCR significantly improves the existing PCR methods for walking because it uses two specific primers, suggesting that higher feasibility and efficiency of IPCR than LMPCR in identify the genomic flanking sequence of D.salina actin gene.

关 键 词：反向PCR 连接介导法PCR 盐藻 肌动蛋白 旁侧序列cDNA 简并引物 

分 类 号：Q754[生物学—分子生物学]