Genome walking by Klenow polymerase

Abstract

Genome walking procedures are all based on a final polymerase chain reaction amplification, regardless of the strategy employed for the synthesis of the substrate molecule. Here we report a modification of an already established genome walking strategy in which a single-strand DNA substrate is obtained by primer extension driven by Klenow polymerase and which results suitable for the direct sequencing of complex eukaryotic genomes. The efficacy of the method is demonstrated by the identification of nucleotide sequences in the case of two gene families (chiA and P1) in the genomes of several maize species.