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

• Main Authors: Wibhasiri Srisuwan, Thanusak Tatu
• Format: Journal
• Published: 2018
• Subjects: Biochemistry, Genetics and Molecular Biology; Medicine
• Online Access: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85054478624&origin=inward; http://cmuir.cmu.ac.th/jspui/handle/6653943832/62591
• Institution: Chiang Mai University

© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group. 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 α- and β-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, αCSα), 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 1 μL of EDTA whole blood and with addition of 10 heat-cool steps (3 min. at 94 °C, followed by 3 min. at 55 °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.