DNA amplification fingerprinting using two long primers

摘要

DNA-based techniques have great potential for detecting genetic variability between genotypes. However, each approach generates different molecular markers, so the kind and amount of polymorphism detected and the cost and time required vary among them. In general, a good method for DNA fingerprinting to detect genetic polymorphism must generate a complex pattern of bands per experiment, the results must be reproducible, and, ideally, the procedure should be quick and convenient. The development of PCR-based technologies offers different tools with which to increase the number of molecular genetic markers and the range of polymorphism detected. RAPD (7) and DNA amplification fingerprinting (DAF) (1) are PCR methods based on the amplification of unknown DNA sequences. Because little or no sequence data for the organism are necessary, these methods can be applied to any species. RAPDs have been widely used, but they produce few bands per primer reaction and their reproducibility is questionable. Other methods have been used to produce DNA fingerprints that are generated by the same mechanism as RAPDs or DAF, the difference being the primer's non-arbitrary sequence. Gillings and Holley (4) described the long primers RAPD technique (LP-RAPD), which is based on the use of long primers' 18-24-mers, designed using the consensus sequence of several families of short interspersed repetitive elements in eubacteria.