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| Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L. |
| Muhammad Abubakkar Azmat, Iqrar Ahmad Khan, Hafiza Masooma Naseer Cheema, Ishtiaq Ahmad Rajwana, Ahmad Sattar Khan, Asif Ali Khan |
| Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan; Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan; University College of Agriculture, Bahauddin Zakariya University, Multan, Pakistan; Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan |
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Abstract Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950‒1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.
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Received: 18 June 2011
Published: 06 April 2012
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