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

Journal article

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 i...

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Authors: Zhang, Z; Gurr, S

• Publication status: Published
• Journal: Gene
• Volume: 253
• Issue: 2
• Pages: 145-150
• Publication date: 2000-08-01
• DOI: 10.1016/s0378-1119(00)00289-4
• EISSN: 1879-0038
• ISSN: 0378-1119
• Language: English
• Keywords: Genes, Fungal; DNA, Fungal; Sequence Analysis, DNA; Base Sequence; Molecular Sequence Data; Polymerase Chain Reaction; Chitin Synthase; Ascomycota; Chromosome Walking; Myosins; Sequence Homology, Amino Acid; Amino Acid Sequence; Exons; Introns; Molecular Motor Proteins