Arrayed primer extension: a robust and reliable genotyping platform for the diagnosis of single gene disorders: beta-thalassemia and thiopurine methyltransferase deficiency.
Mutation screenings, which were conventionally carried out individually because of different assay conditions, are usually time consuming and not cost effective. Using microarray technology, simultaneous molecular diagnosis of multiple mutations on a single platform is possible. To evaluate this idea, we developed a DNA chip platform to simultaneously detect 23 mutations of the beta-globin gene and 9 mutations of thiopurine methyltransferase (TPMT) gene based on the principle of arrayed primer extension (APEX). A blinded test consisting of 200 DNA samples with known genotypes was performed to validate this strategy. High genotyping accuracy of 97.3% and 100% for beta-globin and TPMT genes, respectively, were achieved. Further analysis on the fluorescent intensity demonstrated clear separation between the real signal and the background noise, which enabled us to set two cutoff values (V(lower) = 4.0 and V(upper) = 12.0) to determine the genotype quantitatively. Our results showed that APEX is a highly reliable genotyping strategy to detect mutations that cause beta-thalassemia or TPMT enzyme deficiency.
Lu
Yi
Y
Department of Pediatrics, Division of Hematology and Oncology, National University of Singapore, Singapore.
Kham
Shirley Kow-Yin
SK
Tan
Poh-Lin
PL
Quah
Thuan-Chong
TC
Heng
Chew-Kiat
CK
Yeoh
Allen Eng-Juh
AE
D001483:Base Sequence; D017931:DNA Primers; D005453:Fluorescence; D030342:Genetic Diseases, Inborn / Q000235:genetics*; D006801:Humans; D008780:Methyltransferases / Q000172:deficiency / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017086:beta-Thalassemia / Q000235:genetics*
D017931:DNA Primers; D008780:Methyltransferases; C022745:thiopurine methyltransferase