Cloning and analysis of the 5’ and 3’ flanking regions of the Crinum asiaticum agglutinin gene by genomic walking

Abstract

We reported a simple and efficient method, which combines restriction endonuclease digestion and adaptor ligation, for cloning unknown genomic sequences adjacent to a known sequence. After total
genomic DNA is completely digested with the different sticky-end restriction endonuclease separately, the ends are full. The DNA fragments with blunt-end were then ligated separately to the adaptor. The adaptor-ligated genomic DNA fragments are used as template for cloning flanking regions from all sequence of interest. A first round PCR is performed with a gene-specific primer and the adaptor primer at its 5’ and 3’ end. This is followed by second PCR amplification with a nested gene-specific primer and the nested adaptor primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the 5’ and 3’ flanking region of a mannose-binding lectin gene based upon DNA fragment obtained from China Crinum (Crinum asiaticum var. (Roxb. ex Herb.) Barker).