Hybridization analysis of DNA blots.
The principle of hybridization analysis is that a single-stranded DNA or RNA molecule of defined sequence (the probe) can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the target), with the stability of the hybrid depending on the extent of base pairing that occurs. Experimentally, the analysis is usually carried out with a probe that has been labeled and target DNA that has been immobilized on a membrane support. Hybridization analysis is sensitive and permits detection of single-copy genes in complex genomes. This unit presents a basic procedure for hybridization analysis with a radiolabeled DNA probe. Despite its lack of embellishment, the protocol gives acceptable results with Southern blots on nitrocellulose and nylon (uncharged and charged) membranes. An Alternate Protocol describes a similar method for probing DNA blots with a radiolabeled RNA probe. A Support Protocol for stripping blots to ready them for reprobing is also provided.
Brown
T
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University of Manchester Institute of Science and Technology, Manchester, United Kingdom.
D000818:Animals; D026501:Blotting, Southwestern / Q000295:instrumentation / Q000379:methods*; D002874:Chromosome Mapping / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D018628:Gene Dosage; D016678:Genome; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012313:RNA / Q000032:analysis / Q000737:chemistry
D012313:RNA; D004247:DNA