Multiplexed Analysis Using Time-Resolved Near-IR Fluorescence for the Detection of Genomic Material

While fluorescence continues to be an important tool in genomics, new challenges are being encountered due to increased efforts toward miniaturization reducing detection volumes and the need for screening multiple targets simultaneously. We have initiated work on developing time-resolved near-IR fluorescence as an additional tool for the multiplexed analyses of DNA, either for sequencing or mutation detection. We have fabricated simple and compact time-resolved fluorescence microscopes for reading fluorescence from electrophoresis or DNA microarrays. These microscopes consist of solid-state diode lasers and diode detectors (SPADs) and due to their compact size, the optical components and laser head can be mounted on high-speed micro-translational stages to read fluorescence from either multi-channel capillary electrophoresis instruments or microfabricated DNA sorting devices. The detector is configured in a time-correlated single photon counting format to allow acquisition of fluorescence lifetimes on-the-fly during data acquisition in the limit of low counting statistics. In multiplexed analyses, lifetime discrimination serves as a method for dye-reporter identification using only a single readout channel. Also, coupled to multi-color systems, lifetime identification can significantly increase the number of probes monitored in a single instrument. In this work, near-IR fluorescence, including dye-labels and hardware, will be discussed as well as the implementation of near-IR fluorescence in DNA sequencing using slab gel electrophoresis and DNA microarrays.