DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors

被引:11
作者
Cardozo, Daniela Maira [1 ]
Guelsin, Glaucia Andreia [1 ]
Clementino, Samaia Laface [1 ]
de Melo, Fabiano Cavalcante [1 ]
Braga, Marco Antonio [1 ]
de Souza, Cleonice [2 ]
Moliterno, Ricardo Alberto [1 ]
Laguila Visentainer, Jeane Eliete [1 ]
机构
[1] Univ Estadual Maringa, Dept Anal Clin, Lab Imunogenet, BR-87020900 Maringa, PR, Brazil
[2] Secretatia Saude Estado Parana, Maringa, PR, Brazil
关键词
DNA extraction; Coagulated blood; Molecular biology; Standardization; CLOTTED HUMAN BLOOD;
D O I
10.1590/S0037-86822009000600008
中图分类号
R38 [医学寄生虫学]; Q [生物科学];
学科分类号
07 ; 0710 ; 09 ; 100103 ;
摘要
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 (R) commercial kit (Biological Industries, Beit Haemek, Israel), die Neoscience (R) 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/mu l), which were measured using die Qubit (R) 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/mu l). 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.
引用
收藏
页码:651 / 656
页数:6
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