Optimized RAPD analysis generates high-quality genomic DNA profiles at high annealing temperature.

Techniques such as arbitrarily primed polymerase chain reaction (APPCR) (11), random amplified polymorphic DNA (RAPD) (12) and DNA amplification fingerprinting (DAF) (7) are powerful tools for genetic mapping, taxonomic and phylogenetic investigations and for the detection of various types of DNA damage and mutation (2,3). Nevertheless, the arbitrary nature of the DNA polymerization catalyzed in these methods has drawn criticism because of the low annealing temperature used with short primers. In this study, the RAPD method was optimized at an annealing temperature of 50°C for 10-mer primers. The main advantage of high stringency conditions is that nonspecific reactions are significantly reduced. Thus, protocols that use a high annealing temperature should be always preferred if the sensitivity of polymorphism detection is not jeopardized. Although DNA profiles have been generated at a high annealing temperature for short primers (5,6,8), an optimization study using high annealing temperature has rarely been attempted, to our knowledge. Indeed, it is generally believed that repeating a PCR assay is jeopardized by annealing temperatures higher than the Td of the oligonucleotide primer. For instance, it has been reported that annealing temperatures above 40°C prevent amplification by 10-mer primers (12). Relatively low annealing temperatures (34°C–36°C) are used in RAPD to ensure a maximal number of primer binding events and the consequent generation of many amplified DNA fragments for analytical purposes. However, the low stringency of the accompanying DNA hybridization can result in the formation of spurious amplifications (9) that affect both the reproducibility (8) and the detection of Mendelian inheritance patterns (4). The RAPD method was developed as a result of the sequential and systematic analysis of the RAPD protocol performed by the variation of annealing temperatures, DNA purity, primer sequence and the relative concentration of each PCR reagent. The objective was to generate reproducible DNA profiles of high discrimination with a maximum number of bands, good product yield and clarity, while reducing the occurrence of spurious amplifications in the negative control reactions. Initially, the parthenogenetically produced offspring of Daphnia magna (clone 5) (2) was chosen to eliminate the possibility of confusing genomic changes from sexual reproduction. The optimization work was also performed using other species belonging to the bacteria, plant and animal kingdoms. Table 1 shows the results of the optimization study. Contrary to RAPD methods using low annealing temperatures, consistent genomic profiles were generated when component concentrations were subjected to variation, within the predefined optimal conditions. DNA of good purity and free from other macromolecules and inhibitory compounds produces clear and discriminatory RAPD profiles. For the bulk of our experiments, genomic DNA prepared by a standard phenol/chloroform extraction purification was sufficient to produce RAPD profiles of high quality; profiles of identical quality were obtained using either phenol chloroform or caesium chloride extracted DNA. Further experiments also confirmed that the storage buffer (analytical grade water or 1× Tris-Borate-EDTA buffer) had no influence on RAPD profiles. In addition, under optimized conditions, identical RAPD profiles were generated whenever the DNA extraction or PCR was performed. The DNA concentration was also found to be crucial in the production of reproducible genomic profiles, not only to ensure the largest number of amplified bands but also to Benchmarks