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 6x1 microg of genomic DNA (ca 20kb 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 6kb 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, BgChs2, which includes a myosin motor-like sequence fused to a type V chitin synthase gene.
Zhang
Z
Z
Department of Plant Sciences, University of Oxford, South Parks Road, OX1 3RB, Oxford, UK.
Gurr
S J
SJ
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002687:Chitin Synthase / Q000235:genetics; D016386:Chromosome Walking; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D005091:Exons; D005800:Genes, Fungal / Q000235:genetics; D007438:Introns; D020409:Molecular Motor Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009218:Myosins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D020409:Molecular Motor Proteins; D002687:Chitin Synthase; D009218:Myosins