Open AccessThe reproducibility of RAPD profiles: Effects of PCR components on RAPD analysis of four centaurium species
Author(s) -
Marijana Škorić,
Branislav Šiler,
Tijana Banjanac,
Jasmina Zivkovic,
Slavica Dmitrović,
Danijela Mišić,
D. Grubišić
Publication year - 2012
Publication title -
archives of biological sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.217
H-Index - 25
eISSN - 1821-4339
pISSN - 0354-4664
DOI - 10.2298/abs1201191s
Subject(s) - rapd , primer (cosmetics) , biology , polymerase chain reaction , dna , microbiology and biotechnology , reproducibility , genetic diversity , genetics , chromatography , chemistry , gene , population , medicine , environmental health , organic chemistry
Random amplified polymorphic DNA (RAPD) analysis is a simple and reliable method used to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of the assay. In this study, we analyzed the effects of different concentrations of primer, magnesium chloride, template DNA and Taq DNA polymerase to develop and standardize a RAPD protocol for Centaurium species. The optimized PCR reaction mixture included: 50 ng of DNA extracted using a CTbased protocol, 2.5 mM MgCl2, 7.5 pmol primer and 2 U of Taq polymerase in a final volume of 25 μl. Each of the five primers used in experiments (OPB11, OPB15, OPB18, OPF05 and OPH02) generated reproducible and distinguishable fingerprinting patterns of four Centaurium species. The obtained optimized RAPD protocol and the selected primers are useful for our further work in the genetic diversity studies of Centaurium species
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