Molecular analysis of unknown beta-globin gene mutations using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique and its application in Thai families with beta-thalassemias and beta-globin variants.

摘要

OBJECTIVES: Approximately 40 beta-globin gene mutations have been identified in Thailand. The detection of these mutations is currently performed by the reverse dot blot (RDB) hybridization technique, which could detect only known mutations. We describe here the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assay for detecting unknown mutations of the beta-globin genes. DESIGN AND METHODS: Six PCR fragments covering the promoter, entire coding region, and intervening sequences were amplified before separation by the SSCP technique. Fifteen known mutations and two polymorphisms were analyzed by this technique in various gel mixtures and temperatures to compare their mobility shift patterns. RESULTS: The clear patterns of mobility shift were demonstrated when a 10% polyacrylamide gel with 5% glycerol was used. The sensitivity was found to be 100% when electrophoreses were performed at both room temperature and 6 degrees C. This technique was then applied to screen beta-globin gene mutations in Thai families with similar profiles of abnormal hemoglobins. The distinct patterns of mobility shifts were observed in which further sequencing analysis revealed an AC insertion at codon 146, causing hemoglobin Tak. CONCLUSION: The PCR-SSCP technique might be a useful molecular technique to minimize the requirement of direct genomic sequencing to identify beta-globin gene mutations and could be applied in several developing countries where resources are limited but genetic hemoglobin disorders are highly prevalent.