This Phase I SBIR project introduces a novel laser-induced fluorescence (LIF) detector that will raise DNA sequencing to a new level. All commercial sequencers make basecalls from one-dimensional fluorescence data, viz., intensities at four wavelengths. Fluorescence lifetime, another basecalling option, is developmental. Our concept uniquely generates fully two-dimensional data arrays as complete fluorescence decay profiles are measured at several wavelengths simultaneously. The approach relies on a high repetition rate pulsed laser, has no moving parts, and uses a simple photomultiplier tube instead of a CCD camera. Of the many benefits, the most notable is that the two-dimensional data format offers a multiplicative improvement in basecalling accuracy. If the probability of an inaccurate basecall is 5 percent for spectral data and 10 percent for lifetime data, the combined data reduces the inaccuracy to 0.5 percent. Moreover, the ability to resolve overlapping peaks and discriminate against background fluorescence improves dramatically, allowing far longer fragments to be sequenced in much shorter time. Projected performance by the end of Phase II includes ability to measure in parallel on 16 capillaries, read length greater than 1000 bases with better than 99.5 percent basecalling accuracy, and less than 90-minute run time. No commercial instrument comes close to this combination of speed and accuracy.
Automated DNA sequencers have revolutionized genetic research and diagnostic procedures. Annual sales of $200 million were estimated for automated DNA sequencing equipment in 1999 and the size of the market is expected to increase sharply during the next five years. The need for an automated DNA sequencer that can provide more accurate basecalling, longer real lengths, and shorter run times will be met by the proposed research, in which the first two-dimensional fluorescence detector for sequencing will be developed.
