An efficient and rapid method for gene cloning from eukaryotic genomic DNA using overlap-PCR: With an example of cattle Ghrelin gene.
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.
Zhang
Ai-Ling
AL
College of Animal Science and Technology, Northwest A&F University, Yang-ling 712100, People's Republic of China.
Zhang
Li
L
Zhang
Liang-Zhi
LZ
Chen
Hong
H
Lan
Xian-Yong
XY
Zhang
Chun-Lei
CL
Zhang
Cun-Fang
CF
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005091:Exons / Q000235:genetics*; D016678:Genome / Q000235:genetics; D054439:Ghrelin / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D054439:Ghrelin