A Simple Whole-Blood Polymerase Chain Reaction without DNA Extraction for Thalassemia Diagnosis

被引:1
作者
Srisuwan, Wibhasiri [1 ]
Tatu, Thanusak [1 ]
机构
[1] Chiang Mai Univ, Fac Associated Med Sci, Div Clin Microscopy, Dept Med Technol,Res Ctr Hematol & Hlth Technol, Chiang Mai, Thailand
关键词
Betaine; DNA extraction; polymerase chain reaction (PCR); thalassemia; whole-blood; ALPHA-THALASSEMIA; HEMOGLOBIN-E; PCR; AMPLIFICATION; PURIFICATION; BETAINE;
D O I
10.1080/03630269.2018.1496929
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Polymerase chain reaction (PCR) diagnosis of thalassemia usually relies on using genomic DNA. Preparing the genomic DNA can lead to sample-to-sample contamination. This report was aimed to establish the PCR protocol using whole-blood for detecting mutations of - and -globin genes causing the thalassemia syndrome. First, the PCR facilitators, betaine and bovine serum albumin (BSA), were tested, simultaneously with an adjustment of PCR thermal cycler and of whole-blood volume. Thereafter, the established whole-blood PCR was applied for detecting, in both known and unknown samples, the HBA1 Southeast Asian (--(SEA)) (NG_000006.1: g.26264_45564del19301) deletion, Hb Constant Spring (Hb CS, HBA2: c.427T>C, (CS)), codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) deletion, -28 (A>G) (HBB: c.-78A>G) and codon 26 (G>A) (Hb E or HBB: c.79G>A). It was shown that the whole-blood PCR worked successfully in 9.0% (w/v) betaine, with 1L of EDTA whole blood and with addition of 10 heat-cool steps (3min. at 94 degrees C, followed by 3min. at 55 degrees C) prior to the typical thermal cycles for the mutations. The capability of the new whole-blood PCR was similar to that of the typical DNA-based PCR. Therefore, the newly established whole-blood PCR could be performed for PCR diagnosis of thalassemia. Using this platform, sample-to-sample contamination should be eliminated.
引用
收藏
页码:178 / 183
页数:6
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