Single Quantum Dot-based Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

We propose an assay which combines the specificity of gap-filling ligation chain reaction (Gap-LCR) and the sensitivity of quantum dot-single molecule spectroscopy (QD-SMS) to detect point mutation from genomic DNA without pre-PCR amplification. Mutation variant-specific ligation products are generated by Gap-LCR and captured by quantum dot to form DNA-QD nanocomplexes that are detected by SMS. SMS enables measurement of fluorescent bursts emitted from individual molecules, eliminating the need of separation of ligation products. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants and allows for detection of multiple types of mutation variants from a single experiment.