title: [DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors].
abstract: The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience column kit (One Lambda Inc., San Diego, CA, USA) and a modified salting-out method. Only the salting-out method was able to extract high concentrations of DNA (mean, 180 ng/(1/4)microl), which were measured using the Qubit fluorescence detector (Invitrogen, USA). This method enabled amplification of HLA (human leukocyte antigen) genes using the Luminex PCR-SSO (polymerase chain reaction - sequence-specific oligonucleotide) technology, which demands good quality DNA, and amplification of KIR (killer-cell immunoglobulin-like receptor) genes using an in-house PCR-SSP (polymerase chain reaction - sequence-specific primer) technique, which demands a specific concentration of DNA (10 ng/(1/4)microl). We concluded that the modified salting-out technique was very efficient, simple and fast for DNA extraction from human coagulated blood samples, with the aim of genotyping the HLA and KIR genes.
authors.affiliation: Laboratório de Imunogenética, Departamento de Análises Clínicas, Universidade Estadual de Maringá, Maringá, PR. jelvisentainer@uem.br
authors.forename: Daniela Maira
authors.initials: DM
authors.lastname: Cardozo
authors.forename: Gláucia Andréia
authors.initials: GA
authors.lastname: Guelsin
authors.forename: Samaia Laface
authors.initials: SL
authors.lastname: Clementino
authors.forename: Fabiano Cavalcante de
authors.initials: FC
authors.lastname: Melo
authors.forename: Marco Antônio
authors.initials: MA
authors.lastname: Braga
authors.forename: Cleonice de
authors.initials: Cd
authors.lastname: Souza
authors.forename: Ricardo Alberto
authors.initials: RA
authors.lastname: Moliterno
authors.forename: Jeane Eliete Laguila
authors.initials: JE
authors.lastname: Visentainer
mesh_terms: D004247:DNA / Q000097:blood / Q000302:isolation & purification*; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction / Q000379:methods; D054340:Receptors, KIR / Q000235:genetics*
chemical_list: D006680:HLA Antigens; D054340:Receptors, KIR; D004247:DNA