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An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene |
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| Authors: | Ai-ling Zhang Li Zhang Liang-zhi Zhang Xian-yong Lan Cun-fang Zhang |
| Affiliation: | a College of Animal Science and Technology, Northwest A&F University, Yang-ling 712100, People’s Republic of China b College of Animal Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China c Agricultural College, Guangdong Ocean University, Zhanjiang 524000, People’s Republic of China d College of Lifesciences, Xuzhou Normal University, Xuzhou 221000, People’s Republic of China |
| Abstract: | 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. |
| Keywords: | Overlap-PCR Megeprimer Gene cloning Genomic DNA Gene splicing Cattle Ghrelin gene |
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