title: Optimization of DNA extraction for RAPD and ISSR analysis of Arbutus unedo L. Leaves.
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 A(260)/A(280), 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.
authors: Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Campus St Apolónia, Apartado 1172, 5301-855, Bragança, Portugal; E-Mails: olga_de_sa@hotmail.com (O.S.); jpereira@ipb.pt (J.A.P.).
authors: Olga
authors: O
authors: Sá
authors: José Alberto
authors: JA
authors: Pereira
authors: Paula
authors: P
authors: Baptista
mesh_terms: D002593:Cetrimonium Compounds / Q000737:chemistry; D018744:DNA, Plant / Q000032:analysis* / Q000302:isolation & purification; D004229:Dithiothreitol / Q000737:chemistry; D029791:Ericaceae / Q000235:genetics*; D008623:Mercaptoethanol / Q000737:chemistry; D018895:Microsatellite Repeats / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D011205:Povidone / Q000737:chemistry; D019105:Random Amplified Polymorphic DNA Technique; D013696:Temperature
chemical_list: D002593:Cetrimonium Compounds; D018744:DNA, Plant; D008623:Mercaptoethanol; D011205:Povidone; D004229:Dithiothreitol