A specific and versatile genome walking technique.

We describe here a nested PCR-based strategy for genome walking to extend a known sequence region to its uncharacterized flanking regions. This technique involves the use of a partially degenerate primer as a walker primer and a set of nested specific primers to perform two to three successive rounds of nested PCR. To increase the success rate of genome walking, four different walker primers were designed to allow the setup of parallel reactions. This technique was applied to amplify flanking sequences of known genomic loci of two highly divergent photosynthetic organisms, Rhodobacter capsulatus and Heliophilum fasciatum. Specific products were preferentially amplified using this strategy, which were verified using DNA sequencing. The extremely high success rate of extension of genomic regions in these two organisms suggests that this technique can be applied to a wide range of genomes.