title: Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation.
abstract: The reproducibility and potential applications of anonymous amplification protocols can be improved by using pairs of primers, each of 18 to 24 bases, to replace the single 8 to 10 base primers normally used in randomly amplified polymorphic DNA (RAPD) or DNA amplification fingerprinting (DAF) methods. Amplification using large primer pairs (LP-RAPD) generates 5 to 30 bands that can be resolved on standard agarose gels. Complex fingerprints can be readily generated from viruses, bacteria, fungi, plants, invertebrates and vertebrates. We also present evidence that a number of polymerase chain reaction (PCR) methods, including those based on the use of enterobacterial repetitive intergenic consensus (ERIC-PCR) or microsatellite primed (MP-PCR) sequence, may in essence operate by the same mechanism as LP-RAPD. Using standard LP-RAPD protocols, reproducible fingerprints can be generated from a single specimen using different thermocyclers, regardless of the mechanism used for thermocycling (air-cooled, Peltier effect, or robotic arm). LP-RAPD is sensitive to intraspecific and interspecific genetic variation, demonstrated here by analysis of mites and apple cultivars. Approximately 50% of LP-RAPD products are expected to have different primers at either end. Polymorphic bands with this arrangement can be recovered from the gel and directly sequenced using the LP-RAPD primers themselves. The efficiency of sequencing is improved by the length of the LP-RAPD primers. This method has the potential to allow the production of allele-specific species markers in less than two days.
authors.affiliation: Key Centre for Biodiversity and Bioresources, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia. mgilling@rna.bio.mq.edu.au
authors.forename: M
authors.initials: M
authors.lastname: Gillings
authors.forename: M
authors.initials: M
authors.lastname: Holley
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis*; D016172:DNA Fingerprinting; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004271:DNA, Fungal / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D005784:Gene Amplification; D014644:Genetic Variation; D018598:Minisatellite Repeats; D019105:Random Amplified Polymorphic DNA Technique; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004271:DNA, Fungal; D004279:DNA, Viral; D004247:DNA