Single nucleotide polymorphism discovery of with chromosome walking PCR method

Abstract

In this paper, the basic principle of chromosome walking is presented and we used an actin gene of radiata pine (Pinus radiata) as an example to conduct upstream and downstream chromosome walking for EST sequences. The full genomic sequence (2154 bp) of the actin gene, including promoters 5′ UTR, CDS and 3′ UTR, was identified by chromosome walking. PCR amplification and DNA band sequencing from 200 unrelated radiata pine trees revealed a total of 21 SNPs for the actin gene, three in the promoter region, 15 in CDS and 4 in 3′ UTR. The results of this experiment provide a technical framework for SNPs discovery in none coding regions of candidate genes.

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