There is a great deal of interest in the development of automated DNA mapping and sequencing methodologies which is particularly important, in view of the recent interest in sequencing the human genome. The successful completion of this ambitious project will require improved automation. Sequence data are presently being added to data banks at a rate of 10.sup.6 bases/year but the human genome contains 3.times.10.sup.9 base pairs.
The detection system presently used by most workers in DNA sequencing or mapping involves using radioisotope labeled DNA. The radioactive slab gels in which the DNA fragments have been separated are placed against an x-ray film for overnight exposure of the film. After the exposure and development of the x-ray film, the sequence or size of the DNA separated fragments are read directly from the images on the film.
The autoradiographic detection method described above is not only slow but also requires handling and disposal of hazardous radioactive materials. The reason autoradiography is still so widely used is because it uniquely provides the necessary sensitivity.
There has been great interest in automating the sequence determination procedures using recent advances in optical, electronic and computer technology. Autoradiographic films can now be digitized by a scanning transmission densitometer or video cameras and the digitized images can be computer processed to determine DNA sequences. These digitizing and automated sequence determination systems use autoradiography as the primary detection method.
In 1986, L. M. Smith, J. Z. Sanders, R. J. Kaiser, P. Hughes, C. Dodd, C. R. Connell, C. Heiner, S. B. H. Kent and L. E. Hood, Nature, vol. 321, pp. 674-679, developed a method for detecting fluorescently labeled DNA on gels which they believe is capable of sequencing .about.15,000 base pairs per day. They state that one of the three areas needing development is "increasing the detection sensitivity of the system thereby allowing less material to be used per reaction which in turn allows the use of thinner gels having higher resolution." An apparatus developed by W. Ansorge, A. Rosenthal, B. Sproat, C. Schwager, J. Stegemann, and H. Voss, Nuc. Acids Res., vol. 16, pp. 2203-2207 (1988), using a slightly different protocol, was able to sequence 500 base pairs in 5 hours with a sensitivity per band of 10.sup.-18 mole or 6.times.10.sup.5 molecules. An analogous approach with similar capabilities was developed by J. M. Prober, G. L. Trainor, R. J. Dam, F. W. Hobbs, C. W. Robertson, R. J. Zagursky, A. J. Cocuzza, M. A. Jensen and K. Baumeister, Science, vol. 238, pp. 336-341 (1987).
The development of a high sensitivity detection system would obviously be very important. If very small amounts of fluorescence labeled DNA can be detected on gels, then less labeled DNA is required and the thickness of the gel can be reduced. A thinner gel will have higher resolution so it will not have to be run out as far to resolve the bands. This could result in a major saving in time. Also, the available fluorescence DNA sequencing systems require a detection system that is dedicated to the electrophoresis system during the entire approximate 10 hour run. The detection system would be more efficiently used if it detected the gels off-line from the electrophoresis. This could also result in a major saving of time and increase in throughput.
USPTO Backgrounds
USPTO Backgrounds