Detection and haplotype differentiation of Southeast Asian alpha-thalassemia using polymerase chain reaction and a piezoelectric biosensor immobilized with a single oligonucleotide probe.

DNA-based diagnosis of alpha-thalassemias routinely relies on polymerase chain reaction (PCR) and gel electrophoresis. Here, we developed a new procedure for the detection and haplotype differentiation of Southeast Asian (SEA) alpha-thalassemia using a 3-primer system for PCR coupling with a DNA-based piezoelectric biosensor. PCR products amplified from genomic DNA were differentiated directly by using a quartz crystal microbalance immobilized with a single oligonucleotide probe. The frequency changes after hybridization of the PCR products amplified from a representative sample of normal alpha-globin, SEA alpha-thalassemia heterozygote, and homozygote were 206+/-11, 256+/-5, and 307+/-3 Hz, respectively. The fabricated biosensor was evaluated through an examination of 18 blind specimens. It could accurately discriminate between normal and SEA alpha-thalassemic samples, which suggests that this biosensor system is a promising alternative technique to detect SEA alpha-thalassemia because of its specificity and less hazardous exposure as compared with conventional methods.