Fidelity genotyping of point mutation by enhanced melting point difference using DNA ligase

Melting curve analysis is a powerful tool for detecting single-base mutations that may be linked to genetic diseases. Current existing methods provide insignificant melting point difference for some point mutations with the risk of wrong genotyping results, causing great limitations to their applications in clinic diagnosis. Here, we have developed an enhanced melting point difference approach to genotype single-base mutations using DNA ligase. Ligase covalently joins an allele-specific discriminating probe and a signal probe flanked the mutation site to form a long duplex, resulting in an enhanced melting temperature. CD17 and Ivs-2-654 point mutations of beta-globin gene in thalassemia disease were identified by using this approach, and the homozygotes and heterozygotes were scored accurately and conveniently. To the best of our knowledge, the use of ligase to improve the differences of melting temperature between various genotypes has not been reported. This method will provide a promising tool for clinical diagnosis of gene-mutant diseases.