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 tedious assay processes. In this report, we propose an assay technology which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single molecule coincidence detection and 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.

Quantum dots and single molecule detection enable highly sensitive analysis of genetic biomarkers

“…Since the adventure of SMD, this technology has greatly advanced fundamental study in molecular bindings and interactions. Recently, by incorporating new probes or probe strategies, such as fluorescence resonance energy transfer (FRET)-based probes and dual-color fluorescence coincidence analysis, SMD has been utilized in genomic analysis including detection of DNA of specific sequences and genetic variations, such as single-nucleotide polymorphisms (SNPs) [7,8].…”

Nanotechnologies in Protein Microarrays

“…By engineering the band-gap of QDs (by changing their size), they can emit light of different wavelengths (from the ultraviolet to the near infra-red region), having broad and diverse applications [92]. QDs Fluorescence resonance energy transfer (FRET)-based nanosensors mediated the ultrasensitive detection of low concentrations of target DNA in the diagnosis of genetic diseases were applied [93]. QDs are also used for labeling of peptides, proteins or oligonucleotides as an attractive alternative of traditional organic dyes.…”

Quantum dots in diagnostics and detection: principles and paradigms

“…Figure 6 (C) shows how the encoding scheme might be used with single-bead spectroscopy for a highly multiplexed assay. This and other multiplexing schemes have been developed and employed in a number of notable publications, including SNP analysis 51 , single DNA molecule detection via multiplexed color colocalization 31 , genetic mutation analysis using fluorescence coincidence detection 52 and magnetic-QD multiplexed gene expression analysis 53 .…”