SINGLE QUANTUM DOTBASED MULTIPLEXED （单量子DOTBASED多路复用）.pdf

SINGLE QUANTUM DOT-BASED MULTIPLEXED POINT MUTATION DETECTION BY GAP LIGASE CHAIN REACTION 1 1,2 1,2,3,* Yunke Song , Yi Zhang and Tza-Huei Wang 1Department of Biomedical Engineering, 2Sidney Kimmel Comprehensive Cancer Center, 3Department of Mechanical Engineering and Center of Cancer Nanotechnology Excellence, The Johns Hopkins University, USA ABSTRACT 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 amplifica- tion. 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. KEYWORDS: Gap Ligase Chain Reaction, Quantum Dot, Single Molecule Spectroscopy, Mutation Detection INTRODUCTION Point mutation is a type of widely found genetic aberrations that serve as surrogate biomarkers with high prognostic and 1 diagnostic values . Current point mutation detection techniques heavily rely on PCR amplification of genomic DNA hence are complicated by difficulties associated with PCR, such as non-specific amplifications and limited multiplexing. We report a PCR-independent method capable of multiplexed detection of point mutations from crude genomic DNA samples. This meth- od combines the high specificity of Gap-LCR2 to allow