A new scheme is proposed for detection of electrophoretically separated, fluorescent-labelled DNA fragments for genome sequencing, in which fluorescence lifetime replaces spectral selectivity for distinguishing between the four base-specific labels. Thus, the scheme is based on detection of four fluorescence decays instead of four colors in a single- lane separation of the DNA fragments. In contrast to the analog nature of intensity (""""""""color"""""""") detection, fluorescence lifetime is a concentration- independent characteristic that provides digital detection, offering important advantages for chromatographic detection. Moreover, since the fluorescence lifetimes of the four dyes will be known, data analysis will be simple and straightforward even when there is incomplete chromatographic resolution. Continuous, on-the-fly fluorescence lifetime detection will be performed in the frequency-domain using a state-of-the-art, commercial instrument that incorporates multiharmonic Fourier transform technology to allow measurements of phase and modulation simultaneously at many modulation frequencies. Fluorescence intensity is simultaneously derived from the same measurement to produce a lifetime-intensity chromatogram in no more time than is required for a conventional intensity chromatogram such as is used in the four-color detection scheme. In fact, the improved resolution that is expected from lifetime detection should enable faster sequencing since the demands on the chromatographic resolution will be eased. The proposed research will be performed in three phases. First, candidates for the four base-specific dyes must be identified. The dyes should have similar spectral characteristics and resolvable lifetimes as well as meeting other criteria related to detectability, sensitivity, chemical reactivity and electrophoretic mobility. In addition to visible dyes, primarily of the xanthene group, near-infrared (NIR) dyes will also be investigated. Among the advantages of working in the NIR are low background signal and very low cost instrumentation. Once some promising dyes have been selected, they will be studied individually and in mixtures on electrophoresis gels in order to characterize their spectral and lifetime properties. Limits of detection and lifetime resolution will be determined in order to further narrow down the possibilities, finally producing a few four-dye sets for further investigation. The final phase will be implementation of on-the-fly lifetime detection of labelled DNA, beginning with labelled nucleotides and progressing to short labelled fragments, and eventually progressing to actual sequencing of longer oligonucleotides.
Showing the most recent 10 out of 12 publications
