Plant & Animal Genomes XIII Conference

January 15-19, 2005
Town & Country Convention Center
San Diego, CA

A Genomic Walking Method For The Isolation Of Regulatory Sequences Of Candidate Genes

Siva P. Kumpatla^1,2 , Avutu S. Reddy^1,2

¹  Laboratory for Crop Genome Analysis, Institute for Plant Genomics & Biotechnology, Texas A&M University, College Station, TX 77843, USA

²  Present address: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA

The availability of regulatory sequences that direct constitutive or tissue-specific expression of the genes of interest is crucial to transgenic plant technology. While the conventional methods to isolate such sequences rely on multiple, often time consuming, screenings of genomic DNA libraries using candidate cDNA or other probes, the advent of PCR-based genomic walking techniques facilitated rapid isolation of uncloned genomic DNA sequences. Using a virtual subtraction procedure we have isolated several candidate cDNA clones corresponding to genes that expressed constitutively or spatially. However, when a PCR-based genomic walking method was attempted to isolate the promoter regions of these target genes, candidate sequences were not recovered in the limited number of PCR products obtained. It was later found that the candidate genes were from small multigene families and that we needed to characterize several sequences in order to find the expected regulatory sequences. In order to improve the efficiency of genomic walking, we have developed an alternate method based on magnetic bead capture of target genomic DNA sequences. The capture was followed by a selective PCR using a gene-specific primer and a primer specific to the adaptor ligated to the randomly generated genomic DNA fragments. Using this procedure, we have rapidly obtained several genomic DNA sequences containing the 5’ regulatory sequences of the candidate genes. Details of the method and the results obtained using candidate genes will be presented.