title: Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.
abstract: Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification.
authors.affiliation: Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, 3400 N. Charles Street, Baltimore, MD 21218, USA.
authors.forename: Yunke
authors.initials: Y
authors.lastname: Song
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Tza-Huei
authors.initials: TH
authors.lastname: Wang
mesh_terms: D015374:Biosensing Techniques; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods*; D017354:Point Mutation / Q000235:genetics*; D045663:Quantum Dots