文献类型：期刊文献

英文题名：An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene

作者：Zhang, Ai-ling[1,2];Zhang, Li[1,3];Zhang, Liang-zhi[1];Chen, Hong[1,4];Lan, Xian-yong[1];Zhang, Chun-lei[4];Zhang, Cun-fang[1]

机构：[1]NW A&F Univ, Coll Anim Sci & Technol, Yangling 712100, Peoples R China;[2]S China Agr Univ, Coll Anim Sci, Guangzhou 510642, Guangdong, Peoples R China;[3]Guangdong Ocean Univ, Coll Agr, Zhanjiang 524000, Peoples R China;[4]Xuzhou Normal Univ, Coll Lifesci, Xuzhou 221000, Peoples R China

年份：2010

卷号：391

期号：3

起止页码：1490

外文期刊名：BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

收录：SCI-EXPANDED(收录号：WOS:000274097800035)、、WOS

基金：This study was supported by the National 863 Program of China (Nos. 2006AA10Z197, 2008AA101010), National Natural Science Foundation of China (Nos. 30771544, 30972080), National Key Technology R&D Program (No. 2006BAD01A10-5), Keystone Project of transfergene in China (2009ZX08009-157B, 2008ZX08007-002), "13115" Sci-Tech Innovation Program of Shaanxi Province (2008ZDKG-11), MaTS-Beef Cattle System, Basic and Foreland Technology Study Program of Henan Province (No.072300430160).

语种：英文

外文关键词：Overlap-PCR; Megeprimer; Gene cloning; Genomic DNA; Gene splicing; Cattle Ghrelin gene

外文摘要：In this study, overlap-PCR, an efficient and rapid method, was used to clone cattle Ghrelin gene CDS (coding sequence) from genomic DNA. The procedure included seven primers and three-step PCRs. Cattle Ghrelin gene consists of four exons and the CDS contains 351 bps. In the first step three PCRs were performed to generate extended exon1, exon2, and exon3 that contained overlapped nucleotides and were used as the templates for second ligation PCR. Secondly, exon1 and exon2 were spliced together. And it was same with exon3 and exon4. Lastly, the four exons were linked together with outermost primers and the templates from the second step. Comparison analysis on the obtained CDS of Ghrelin gene and cDNA by RT-PCR showed that the two sequences were same. As an efficient and rapid method, overlap-PCR is feasible and acceptable for gene cloning from genomic DNA. (c) 2009 Elsevier Inc. All rights reserved.