title: Comparative study of DNA extraction methodologies from goat sperm and its effects on polymerase chain reaction analysis.
abstract: Successful DNA extraction is indispensable for molecular methods based on polymerase chain reaction (PCR); however, goat sperm DNA extraction is limited. Thus, the aim of this study was to evaluate three methods to extract DNA from goat sperm for use in PCR. Eight goat semen pools were used for DNA extraction by using the DNeasy Blood & Tissue Kit, phenol-chloroform, and Chelex-100 methods. DNA samples were analyzed spectrophotometrically to determine the DNA concentration and purity, visualized on 0.8% agarose gel, and used at different amounts (150, 100, 50, 10, and 1 ng) for PCR with electrophoresis, followed by 1.5% agarose gel electrophoresis. The quantity of DNA extracted with Chelex-100 was higher (P < 0.05) than that obtained with either the DNeasy Blood & Tissue Kit or the phenol-chloroform method, with the phenol-chloroform method yielding a greater quantity (P < 0.05) than the kit. The DNeasy Blood & Tissue Kit produced a higher (P < 0.05) purity product than the Chelex-100 method, and all samples obtained by the three protocols were positive for DNA, as assessed by electrophoresis. All of the different concentrations of DNA produced by these methods were amplified by PCR, although for DNA produced by the phenol-chloroform method, PCR was only possible after complementary purification. In conclusion, the Chelex-100 method is cheap, secure, simple, fast, and effective, and is a potential tool for extracting goat sperm DNA without limitations in PCR.
authors.affiliation: Laboratório de Andrologia, Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil silva.ecb@gmail.com.
authors.forename: E C B
authors.initials: EC
authors.lastname: Silva
authors.affiliation: Laboratório de Andrologia, Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
authors.forename: M A
authors.initials: MA
authors.lastname: Pelinca
authors.affiliation: Laboratório de Doenças Infecto-Contagiosas, Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
authors.forename: A C
authors.initials: AC
authors.lastname: Acosta
authors.affiliation: Laboratório de Fisiologia e Morfologia Animal, Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
authors.forename: D M F
authors.initials: DM
authors.lastname: Silva
authors.affiliation: Laboratório de Fisiologia e Morfologia Animal, Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
authors.forename: M A
authors.initials: MA
authors.lastname: Gomes Filho
authors.affiliation: Laboratório de Andrologia, Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
authors.forename: M M P
authors.initials: MM
authors.lastname: Guerra
mesh_terms: D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D006041:Goats; D008297:Male; D016133:Polymerase Chain Reaction; D012661:Semen; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D004247:DNA