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Extraction of PCR-usable DNA from trees adapted to arid environment

Author(s): G Sablok, , , and | P Gahlot | A.K.Gupta | K Pareek | N.S.Shekhawat

Journal: Plant Omics
ISSN 1836-0661

Volume: 2;
Issue: 3;
Start page: 103;
Date: 2009;
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Keywords: Prosopis cineraria | Calligonum polygonoides | Acacia nilotica | polyphenols | polysaccharides | DNA extraction | PCR.

Genetic conservation programs in arid natural repertoire rely on molecular methods for diversity assessments. Molecular characterization involves the use of high molecular weight genomic DNA as starting material. Obtaining intact genomic DNA of sufficiently high quality, readily amplifiable using PCR is a primary goal of all molecular genetic studies. The aim of our study was to devise a method for the isolation of the genomic DNA from arid tree species viz. Prosopis cineraria, Calligonum polygonoides and Acacia nilotica (which are rich in polysaccharides, polyphenols and secondary metabolites), hold promises for the restoration of the region. The quality checking of the isolated DNA samples was done through the 10-mer oligonucleotide primers. The present method involves a modification of the available CTAB method employing higher concentration (6%) of polyvinylpyrrolidone (PVP), an extrinsic factor for reducing the metabolite precipitation, higher concentration of CTAB (3%) and an increased period (45 minutes) of incubation with chloroform: isoamylalcohol followed by the RNAse treatment for 90 minutes at 37°C and subsequent pelleting in TE buffer. The yield of DNA ranged from 1240-1500ng/μl and the absorbance lies between 1.7-1.9, indicating minimal levels of contaminating metabolites. The protocol has been tested with three tree species of the arid region which are extremely drought resistant. The DNA isolated was successfully amplified by all the random 10-mer oligonucleotide primers tested with high reproducibility. Optimum annealing temperature was 35°C at a concentration of MgCl2 (1.5mM), lower concentrations of primer (0.2µM) and Taq polymerase (1U). The present method is simple, efficient and economically yielding good quality intact genomic DNA suitable for large genetic screening programs.
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