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Int. J. Mol. Sci. 2011, 12(6), 4156-4164; doi:10.3390/ijms12064156

Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus Stª Apolónia, Apartado 1172, 5301-855, Bragança, Portugal
* Author to whom correspondence should be addressed.
Received: 2 June 2011 / Revised: 16 June 2011 / Accepted: 20 June 2011 / Published: 22 June 2011
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 µg/µL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.
Keywords: Arbutus unedo L.; strawberry tree; DNA isolation; RAPD; ISSR Arbutus unedo L.; strawberry tree; DNA isolation; RAPD; ISSR
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Sá, O.; Pereira, J.A.; Baptista, P. Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves. Int. J. Mol. Sci. 2011, 12, 4156-4164.

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