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Optimization of DNA Extraction Methods from Garcinia species for ISSR-PCR, RAPD-PCR and DNA Barcoding

2016; Volume: 9; Issue: 1 Linguagem: Inglês

10.3923/ajbkr.2017.35.42

1996-0700

Jayesh Anerao, Vikas Jha, Nitin Desai,

Synthesis of Organic Compounds

Background: Genetic analysis and DNA barcoding technology of plant relies on high yields of pure DNA samples.The DNA barcoding has the potential to provide an alternative means of estimating species richness without high level expertise in field identification skills and in a much shorter time frame.Western Ghats of India is one of the mega diversity centres in the world.There are 15 species of Garcinia reported of which many are endemic in nature.Genus Garcinia store large amounts of phenolics, polysaccharides, polyphenols, tannins and other metabolites within their leaf tissue making genomic DNA extraction difficult.While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely mainly on selection of material. Materials and Methods:The DNA of G. indica and G. xanthochymus was extracted by using DNAzol® kit, modified SDS and C-TAB methods.A modified C-TAB and SDS method was optimized for removal of polyphenolics.Results: The protocol developed yielded 2000-2500 ng µLG 1 of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.78-1.81and 260/230 ratio ranging from 2.15-2.18for 3 g of the leaf tissue.The extraction of DNA by SDS was most effective from young leaves.In this study the properties of Triton X in the second purification step to remove lipid and protein component of the cellular membranes has been exploited.Triton X helps in decreasing the surface tension and along with that it helps in solubilization of nonpolar entities.High Concentration of LiCl 2 (0.5 M) in presence of ethanol helps in salting out of DNA.The resulted genomic DNA showed fine Random Amplified Polymorphic DNA (RAPD) Inter Simple Sequence Repeat (ISSR) banding pattern.The DNA obtained was also amenable to rbcL barcode gene amplification of plant.Conclusion: The protocol optimized is reproducible and useful for other members of Garcinia species.