AMPLIFICATION OF ANONYMOUS DNA FRAGMENTS USING PAIRS OF LONG PRIMERS GENERATES REPRODUCIBLE DNA FINGERPRINTS THAT ARE SENSITIVE TO GENETIC VARIATION

摘要

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. [References: 34]