Rapid Isolation of Flanking Genomic DNA Using Biotin-RAGE, a Variation of Single-Sided Polymerase Chain Reaction

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Abstract
A method is described for quickly and reproducibly isolating genomic DNA contiguous with known DNA sequence by means of the polymerase chain reaction (PCR). Flanking genomic DNA is isolated using a biotinylated sequence-specific primer in combination with a generic hybrid primer that binds to a deoxyoligonucleotide sequence artificially added to the ends of the genomic DNA. Amplified sequences that include the biotinylated primer are purified from nonbiotinylated amplification products by binding to a solid-phase streptavidin matrix. The biotinylated amplification product(s) are subjected to a further round of amplification, after which they can be subcloned and analyzed. This technique was applied to the isolation of three intron-exon junctions. Verification of the identity of these junction sequences was accomplished by designing primers based on the intron sequences isolated by Biotin-RAGE, amplifying across the exon using these intron primers, and sequencing the PCR-generated product.