The reproducibility of RAPD profiles: effects of PCR components on RAPD analysis of four Centaurium species

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 concentration...

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Veröffentlicht in:Archives of biological sciences 2012, Vol.64 (1), p.191-199
Hauptverfasser: Skorić, M., Institute for Biological Research Siniša Stanković, Belgrade (Serbia), Šiler, B., Institute for Biological Research Siniša Stanković, Belgrade (Serbia), Banjanac, T., Institute for Biological Research Siniša Stanković, Belgrade (Serbia), Živković, J.N., Institute for Biological Research Siniša Stanković, Belgrade (Serbia), Dmitrović, S., Institute for Biological Research Siniša Stanković, Belgrade (Serbia), Mišić, D., Institute for Biological Research Siniša Stanković, Belgrade (Serbia), Grubišić, D., Institute for Biological Research Siniša Stanković, Belgrade (Serbia)
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Zusammenfassung: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 CTAB based 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.
ISSN:0354-4664
1821-4339
DOI:10.2298/ABS1201191S