Abstract
The genetic studies of tropical tree species containing high amount of phenols are greatly hampered by the inability to extract sufficient quantities of high quality DNA and the presence of PCR inhibitors in extracted DNA samples. While there are some DNA extraction and PCR protocols for such species, no consistency in results. Hence, optimization of such protocols is a prerequisite for conducting research studies on tropical flora. This study reports the optimization of cheaper DNA extraction and PCR procedures for plants having high amount of phenolic compounds and PCR inhibitors. Three fruit crop species, Bael (Aegle marmelos L. Correa), Pomegranate (Punica granatum L.) and Mango (Mangifera indica L.), with distinctly diverse secondary metabolic profiles, were used as examples. The CTAB DNA extraction method, with some modifications, was compared with two commercially available DNA extraction kits namely; Promega Wizard® Genomic DNA purification kit and QIAGEN DNeasy® Plant Mini kit. DNA from three to five genotypes from each species was extracted from each method and the quality and quantity were assessed. Spermidine was added to the PCR mix at the rate of 0.8 μM to block the PCR inhibitors and the DNA samples were amplified using universal plant barcoding primer pair rbcL, and SSR or ISSR primers. The modified CTAB method resulted significantly higher quantity of quality DNA in all samples compared to two commercial kits. Henceforth DNA extracted from CTAB method, and the two commercial kits were used to precede PCR. However, expected bands were not generated in regular PCR. Interestingly, the inclusion of spermidine amplified the relevant band/s in relatively easy PCR reactions such as rbcL, as well as trickier reactions such as SSR and ISSR. These results suggest that cheaper alternative procedures used in this research study could be used successfully for the range of applications in plants with array of secondary metabolic profiles.
Highlights
Optimization of DNA extraction and PCR protocols are the basic and most important preliminary steps of any molecular biological application
Applications of PCR in plant research are unlimited and isolation of high quality DNA is a prerequisite for PCR amplification (Pirttila et al, 2001)
Co-isolation of polysaccharides, polyphenols, RNA and other secondary metabolites interfere with PCR amplification (Demeke and Jenkins, 2010, Sahu et al, 2012)
Summary
Optimization of DNA extraction and PCR protocols are the basic and most important preliminary steps of any molecular biological application Secondary metabolites such as phenolic compounds present in plants play important roles in their defence mechanisms against predators and pathogens (War et al, 2012). These secondary metabolites often hinder the extraction of high quality DNA and successful PCR amplification (Pirttila et al, 2001). There are many DNA extraction methods available from quick and dirty to highly purified column-based commercial kits Most of these methods and commercial extraction kits are optimized for model plant species such as Arabidopsis thaliana and Rice (Oryza sativa) (Roychowdhury et al, 2012). If one protocol works for a range of species, that would save time and cost required for such optimization steps and expedite the genetic improvement of such plant species
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