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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; in revised form: 16 June 2011 / Accepted: 20 June 2011 / Published: 22 June 2011
Abstract: 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
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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.
Sá O, Pereira JA, Baptista P. Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves. International Journal of Molecular Sciences. 2011; 12(6):4156-4164.
Sá, Olga; Pereira, José Alberto; Baptista, Paula. 2011. "Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves." Int. J. Mol. Sci. 12, no. 6: 4156-4164.