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Standardization of DNA Extraction and Optimization of RAPD-PCR Conditions in Garcinia indica

Author(s): Abhijit Sahasrabudhe | Manjushree Deodhar

Journal: International Journal of Botany
ISSN 1811-9700

Volume: 6;
Issue: 3;
Start page: 293;
Date: 2010;
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Keywords: polyphenols and secondary metabolites | DNA isolation | morphological variation | Endemic plant | RAPD conditions

Genetic analysis of plant relies on high yields of pure DNA samples. DNA isolation is difficult in woody plants because of presence of polysaccharides, tannins, alkaloids, polyphenols and other secondary metabolites that interfere during isolation. Here we report for the first time a fast, reliable and less expensive method of genomic DNA isolation from leaves of Garcinia indica. The modified CTAB protocol includes addition of Polyvinylpyrrolidone (PVP) separately in each tube and precipitation with 5M NaCl along with chilled ethanol, which increased the solubility of polysaccharides. Without use of RNase or Proteinase K, were able to isolate pure and sufficient amount of DNA, which proved to be amenable to RAPD analysis. We also optimized RAPD-PCR conditions such as annealing temp, amount of DNA and Taq polymerase etc. A preliminary study of variation within G. indica species was carried out with nine plants with twenty decamers. Out of which six primers showed polymorphism while three had monomorphic banding pattern. In continuation with these promising results, efforts are underway to screen more plants with different geographical locations and with more number of primers to study genetic diversity.
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