New strategies for analyzing molecular signatures of disease states in real time using single pair fluorescence resonance energy transfer (spFRET) were developed to rapidly detect point mutations in unamplified genomic DNA (DNA diagnostics). The assay was carried out using allele-specific primers, which flanked the point mutation in the target gene fragment and were ligated using a thremostable ligase enzyme only when the genomic DNA carried this mutation (ligase detection reaction, LDR). We coupled LDR with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the MB probes formed upon ligation. We demonstrated the ability to rapidly discriminate single base differences in heterogeneous populations having as little as 600 copies of human genomic DNA without PCR amplification. Single base difference in the K-ras gene was discriminated in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA. Real time analyses of point mutations were also performed in PMMA microfluidic device.
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 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 micro fabricated 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.
The intrinsic fluorescence properties of molecules in condensed phases are complicated by intermolecular interactions. In crystalline solids the fluorescence depends on the photophysics of individual molecules as well as the interactions between molecules in the solid state Even a chemically pure crystal has some lattice disorder, which alters the local environment and changes the intermolecular interactions. Sample preparation and experimental technique may influence the fluorescence results.
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