Walking into the unknown: a ‘step down’ PCR-based technique leading to the direct sequence analysis of flanking genomic DNA

We describe a novel and efficient PCR-based technique for walking into unknown flanking genomic DNA without recourse to protracted laborious library screening for overlapping sequences. This two component ‘hot start’ and ‘step down’ PCR method uses 6×1 μg of genomic DNA (ca 20 kb in length) restricted with six different endonucleases and ligated to adaptors with the inclusion of two further restriction enzymes to prevent self-ligation. This allowed us to walk, in a single step, up to 6 kb into flanking DNA and gave sufficient PCR products for up to 200 different walking experiments. This technology enabled us to clone and characterize the previously elusive 5′ sequence of the barley powdery mildew chitin synthase gene, Bg Chs2, which includes a myosin motor-like sequence fused to a type V chitin synthase gene.