A PCR-based method for isolation of genomic DNA flanking a known DNA sequence

• Published in: Current Issues in Molecular Biology Vol. 1; no. 1-2; pp. 47 - 52
• Main Authors: Boulter, C A, Natarajan, D
• Format: Journal Article
• Language: English
• Published: Switzerland 1999
• Subjects: DNA - genetics; DNA - isolation & purification; DNA - metabolism; Gene Library; Genome; Plasmids - genetics; Plasmids - metabolism; Polymerase Chain Reaction - methods
• ISSN: 1467-3037
• DOI: 10.21775/cimb.001.047

We describe a simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of a pUC-based plasmid to generate a limited genomic library. The library is plated onto a number of selective LA plates which are incubated overnight, and recombinant plasmid DNA is then isolated from resistant colonies pooled from each plate. PCR amplification is performed on the pooled recombinant plasmid DNAs using primers specific for the pUC vector and the known genomic sequence. The combination of efficient directional cloning and bacterial transformation gives relative enrichment for the genomic sequence of interest and generates a simple DNA template, enabling easy amplification by PCR.