Point mutation identification using on-chip ligase detection reaction

An efficient method was developed to detect point mutations using oligonucleotide microarrays and the ligase detection reaction (LDR). Allele-specific LDR primers were immobilized on polylysine-coated glass slides to perform LDR on a chip. The spotting concentration and detection limit were analyzed using a synthesized oligonucleotide as a template. The optimal primer spotting concentration was 20 μmol/L, and the lowest detectable template concentration was 0.33 nmol/L. The method was successfully employed to identify malignant mutations of hypertrophic cardiomyopathy. Asymmetric polymerase chain reaction was employed to prepare single stranded DNA as LDR templates from cloned plasmids. The discrimination ratios for AC, TC, GT, TT, GA, and AA mismatches are 32.82, 44.24, 17.75, 18.34, 11.66, and 8.91, respectively. This method may allow construction of multiple mutation detection systems.