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Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction
Author(s) -
Song Yunke,
Zhang Yi,
Wang TzaHuei
Publication year - 2013
Publication title -
small
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.785
H-Index - 236
eISSN - 1613-6829
pISSN - 1613-6810
DOI - 10.1002/smll.201202242
Subject(s) - point mutation , ligase chain reaction , dna ligase , quantum dot , mutation , microbiology and biotechnology , genomic dna , dna , mutant , polymerase chain reaction , genetics , biology , gene , materials science , multiplex polymerase chain reaction , nanotechnology
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.