Chromosomal walking of flanking regions from short known sequences in GC-rich plant genomic DNA

High-efficiency thermal asymmetric interlaced (HE-TAIL) PCR is a modified thermal asymmetric interlaced (TAIL) method for finding unknown genomic DNA sequences adjacent to known sequences in GC-rich plant DNA. Necessary modifications to obtain high-efficiency amplification of flanking sequences are the inclusion of 2 control reactions during tertiary cycling and the design of long gene-specific primers, which can be used during single-step annealing-extension PCR. The modified protocol is suitable to walk from short known sequences, such as sequence-tagged sites (STS), expressed sequence tags (EST), or short exon sequences, and enables researchers to clone full-length open reading frames (ORFs) without library screening. Moreover, the HE-TAIL method can be used to identify DNA sequences flanking T-DNA insertions or to isolate promoter regions. Although individual steps are limited to about 4 kb, multiple steps can be done to walk upstream or downstream of known regions.