A simple and effective method for high quality PCR usable DNA extraction from Azadirachta indica A. Juss
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Authors
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K. Rajarajan
ICAR-Central Agroforestry Research Institute, Jhansi 284 003. U.P., India
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S. Vimala Devi
ICAR-Central Agroforestry Research Institute, Jhansi 284 003. U.P., India
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A.K. Handa
ICAR-Central Agroforestry Research Institute, Jhansi 284 003. U.P., India
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N. Gurunathan
Sub Divisional Forest Officer, Bilaspur Division, Chhattisgarh, India
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S.K. Dhyani
ICAR-Central Agroforestry Research Institute, Jhansi 284 003. U.P., India
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A.R. Uthappa
ICAR-Central Agroforestry Research Institute, Jhansi 284 003. U.P., India
Keywords:
Extraction, genomic DNA, PCR, neem, molecular technique, secondary metabolitesAbstract
Neem is considered as an important tree species because of its valuable wood and biological properties. In general, the extraction of high quality genomic DNA from tree species is difficult due to high phenols, polysaccharides and secondary metabolites. Since high pure DNA is a prerequisite for most of the molecular genetic research, a protocol which does not require the use of Liquid Nitrogen, Walbots method, which was hitherto used only for annual species, was tested for Azadirachta indica, a perennial tree species. The novelty also extends to modifications in this method to make it suitable for species with high polysaccharides, polyphenols and tannins. Major modification includes as addition of (PVP) polyvinyl pyrrolidone 4% in the extraction buffer, increase in the ionic concentration of grinding buffer by increasing NaCl concentration, addition of 0.2% -mercaptoethanol and increased the volume of 20% (SDS) Sodium Dodecyl Sulphate. The genomic DNA obtained by this method was pure and suitable for polymerase chain reaction with RAPD primers. The DNA can be useful for molecular characterization of A. indica.
