DNA Amplification Fingerprinting Using Two Long Primers

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. The aim of this work was to test the capability of two long primers of 19 and 20 nucleotides (F17 and F13, respectively), designed from two hypervariable sequences of rye, Secale cereale L. (6) (GenBank accession nos. F17-AF175285 and F13-AF175284) to amplify DNA from different species and to assess the primer’s capacity to detect genetic variability. The species used in this work included bacteriophage λ, bacteria (two species), yeast, fungi, lichens, plants (10 dicots species and 7 monocots species), and animals (four insects species and two fish species), and mammals (three species including humans). In all plants tested, DNA was isolated by a modification of the method used by Dellaporta et al. (2). PCR amplification was performed in 25 μL containing 50–100 ng DNA, 4 mM MgCl2, 1 μM oligonucleotide primer, 200 μM each dNTP, 1.25 U AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA), and 1× Stoffel fragment buffer (10 mM KCl, 10 mM Tris-HCl, pH 8.3) (Applied Biosystems). The total volume was covered by light mineral oil, and a negative control was included in each experiment to test for possible contamination. Only one primer was used in each reaction. The sequences of the primers were 5′-GAACCAGATTGCTGAGTCC-3′ for F17 and 5′-AAGTGTTGGTTTTGGTTGTG-3′ for F13. After an initial denaturing step of 4 min at 94°C, amplification was carried out over 35 cycles of 1 min at 94°C, 2 min at 55°C, and 5 min at 72°C with a slope of 0.2°C/s, followed by an extension step of 10 min at 72°C. Amplification products were then separated onto 6.5% polyacrylamide gels containing 1× TBE (89 mM Tris-borate, 2 mM EDTA, pH 8.0). The electrophoresis was performed at a constant power of 100 V. The gels were silver stained following the method described by Dias Neto et al. (3). In all cases, we tested the reproducibility of the patterns of bands by comparing the bands of DNA samples from one individual with amplifications performed by different workers and on different PCR repetitions. No differences were noticed between the patterns of bands obtained in each case. Under the amplification conditions used in this work, both primers generated a complex pattern of bands in all the tested organisms (Figure 1). The number of bands generated by F13, per individual, ranged from 18 in bacteriophage λ to 54 in Barbus graellsii, and, in the Benchmarks