[DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors].
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.
Cardozo
Daniela Maira
DM
Laboratório de Imunogenética, Departamento de Análises Clínicas, Universidade Estadual de Maringá, Maringá, PR. jelvisentainer@uem.br
Guelsin
Gláucia Andréia
GA
Clementino
Samaia Laface
SL
Melo
Fabiano Cavalcante de
FC
Braga
Marco Antônio
MA
Souza
Cleonice de
Cd
Moliterno
Ricardo Alberto
RA
Visentainer
Jeane Eliete Laguila
JE
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*
D006680:HLA Antigens; D054340:Receptors, KIR; D004247:DNA