|Vanden ende, Wim|
|Van laere, Andre|
Submitted to: Biotechniques
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/20/2003
Publication Date: 1/20/2003
Citation: Michiels, A., Tucker, M.L., Vanden Ende, W., Van Laere, A. 2003. Chromosomal walking of flanking regions from very short known sequences in gc rich plant genomic DNA. Plant Molecular Biology Reporter. 21:295-302. Interpretive Summary: Understanding how gene expression is regulated is the key to understanding plant growth and development and how plants respond to their environment. To identify regulatory elements in the plant gene that control the induction and level of expression of the gene product it is necessary to clone DNA adjacent to or upstream from DNA that has been previously identified. Very often these upstream regulatory regions are repetitive and rich in one type of nucleotide (DNA building block). We have improved on a procedure commonly used to clone genomic DNA so that it now works better with these types of DNA. These procedural adaptations will be useful information for scientists working to identify uncharacterized DNA that is near to the already characterized DNA sequence of a known gene.
Technical Abstract: The High Efficiency Thermal Asymmetric InterLaced PCR (HE-TAIL) is a modified 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 two 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 like sequence-tagged sites (STS), expressed sequence tags (EST) or short exon sequences and enables researchers to clone full-length ORF without library screening or use of time-consuming 5¿- RACE amplification. Although individual steps are limited to about 4 kb, multiple steps can be done to walk upstream or downstream known regions.