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

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, α^CSα), 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.