Full Length Research Paper

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

Juan Lin¹*, Yuanjie Jin¹, Junhui Wang² and Kexuan Tang¹

¹State Key Laboratory of Genetic Engineering, School of Life Sciences, Morgan-Tan International Center for Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R and D Center, Fudan University, Shanghai 200433, China.

² Research Institute of Forestry, Key Laboratory of Tree Breeding and Cultivation, State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China.

*Corresponding author. E-mail: linjuan@fudan.edu.cn. Tel: +86-21-65642425. Fax: +86-21-65642425.

Abbreviation: GSP, gene specific primer; RE, restriction endonuclease; CAA, Crinum asiaticum agglutinin; AP, adaptor primer; NAP, nest adaptor primer.

Accepted 5 September, 2008

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).

Key words: Crinum asiaticum, Crinum asiaticum agglutinin, genomic walker technology, mannose-binding lectin.