Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

Authors

  • Yunke Song,

    1. Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, 3400 N. Charles Street, 122 Clark, Baltimore, MD 21218, USA
    Search for more papers by this author
  • Yi Zhang,

    1. Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, 3400 N. Charles Street, 122 Clark, Baltimore, MD 21218, USA
    Search for more papers by this author
  • Tza-Huei Wang

    Corresponding author
    1. Department of Mechanical Engineering, Sidney Kimmel Comprehensive Cancer Center and Center of Cancer Nanotechnology Excellence, The Johns Hopkins University, 3400 N. Charles Street, 108 Latrobe, Baltimore, MD 21218, USA
    2. Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, 3400 N. Charles Street, 122 Clark, Baltimore, MD 21218, USA
    • Department of Mechanical Engineering, Sidney Kimmel Comprehensive Cancer Center and Center of Cancer Nanotechnology Excellence, The Johns Hopkins University, 3400 N. Charles Street, 108 Latrobe, Baltimore, MD 21218, USA.
    Search for more papers by this author

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.

Ancillary