Rapid isolation of flanking genomic DNA using biotin-RAGE, a variation of single-sided polymerase chain reaction.
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 identify 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.
Bloomquist
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Neuroscience Department, Johns Hopkins Medical School, Baltimore, MD 21205.
Johnson
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Mains
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D000818:Animals; D001483:Base Sequence; D001710:Biotin; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats
D001710:Biotin; D004247:DNA