RAPD library fingerprinting of bacterial and human DNA: applications in mutation detection.

摘要

Random amplified polymorphic DNA (RAPD) fingerprinting is a modification of the polymerase chain reaction (PCR), which utilises a single, arbitrarily-chosen primer to amplify a number of fragments from a given template DNA to generate a discrete "fingerprint" when resolved by gel electrophoresis. Alterations by as little as a single base in the primer sequence lead to marked alterations in the fingerprints generated with a given template under optimised conditions. By inference, single base alterations in the genomic template DNA may also lead to changes in the RAPD fingerprints. We have examined this potential application to detect mutations in bacteria and cultured human cells. We have utilised Escherichia coli and human lymphoblastoid cell lines exposed to UV radiation, selected for by cellular mutation assays, and compared RAPD fingerprints of mutant and non-mutant samples. Polymorphisms became evident as the presence and/or absence of DNA fragments between the two samples. A dose-dependent increase in the number of polymorphic bands was seen with UV irradiation of E. coli. To a lesser degree, polymorphisms were also evident for human lymphoblastoid DNA. The possible underlying mechanisms for these alterations in fingerprints as a result of mutation(s) in the primer binding site(s) are discussed. The ability of RAPD fingerprinting to detect a mutant in a population of non-mutants is evaluated, and whilst the lack of sensitivity inherent in the technique precludes its use as a mutation screening assay, its potential for generation of mutant and non-mutant DNA probes for other mutation detection techniques may prove to be of great merit. Teratogenesis Carcinog. Mutagen. 20:49-63, 2000. Copyright 2000 Wiley-Liss, Inc.