Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
1999-02-05
2001-04-03
Alexander, Lyle A. (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C436S091000, C436S094000
Reexamination Certificate
active
06210973
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to the investigation of the sequencing of DNA. More particularly, the present invention relates to a method of and apparatus for automating the sequencing of DNA which increases the rate at which DNA can be sequenced as well as improving the reliability and accuracy of the sequencing determination.
DNA sequencing is essential to the practice of biotechnology, genetic engineering and many other disciplines that rely on the need to determine the genetic information contained in DNA. The sequencing of DNA is the process of determining the sequence of nucleic acid bases that comprise a strand of DNA. There are four bases, denoted A for adenine, G for guanine, C for cytosine, and T for thymine, that comprise the DNA. The sequence of these bases uniquely describes each piece of DNA. Sequencing is a crucial step in genetic engineering and biotechnology, since it provides the precise code of genetic information contained in a sample of DNA.
DNA is double stranded and hence, the term base pairs is often used, since each base of one strand is opposed by its complimentary base on the other strand. There are an enormous number of bases that need to be sequenced in order to read a piece of DNA. Even a simple piece of DNA from a bacteria cell would likely comprise several thousand bases. The Human Genome Project, a large, multi-year, United States Government funded national project to sequence the DNA in humans, is attempting to sequence the approximately 10
9
bases found in human DNA.
DNA sequencing is a very labor intensive process and, with the large amounts of DNA that are needed to be sequenced for the biotechnology industry to progress, methods and apparatus to automate this process are very desirable. Much has been written about DNA sequencing and genetic engineering and the reader is referred to the many references in this subject which will provide additional background information.
Two methods of DNA sequencing have been developed. The first is by Maxam and Gilbert, and is described in
Proc. Natl. Acad. Sci. USA
by A. M. Maxam and W. Gilbert, Vol. 74, page 560 (1977). The second method is described in
Proc. Natl. Acad. Sci. USA
, by F. Sangen, S. Nicklen and A. R. Coulson, Vol. 74, page 5463 (1977). Both of those methods involve performing a number of steps -before the fragments of DNA are ready to be detected to yield a sequence. Those steps will not be reviewed here, as they are detailed in the two references noted above. Although the two techniques differ, eventually one arrives at four samples of DNA fragments that end at a given base. For instance, one sample contains fragments that end in base A; another other contains fragments that end in base G, and so forth.
The task is to separate those fragments by size and see what order they are in. If the shortest fragment in all of the four samples is one that ends in T, then the first base of the sequence is T. If the second shortest one ends in C, then the next base in the sequence is C, and so forth, until all of the fragments are separated in order of increasing length and the sequence is determined.
In order to perform this size separation and fragment detection, the first methods of manual DNA sequencing utilized polyacrylamide gel plectrophoresis techniques to separate the fragments. Polyacrylamide gels have the ability to resolve fragments with a resolution of one base pair, and that resolution is necessary for sequencing. Each fragment is labeled with a radioactive element that typically gives off a beta particle, such as radioactive phosphorus, P-32. Each of the four samples are then separated in size in their own lane in the gel. The four lanes are typically side by side. After electrophoresis, a piece of x-ray film is placed next to the gel for a number of hours, often a couple of days, to expose the film with the radioactive emissions from the P-32 phosphorus. When developed, the fragments show up as dark bands on the film and the sequence can then be read from the order in which the bands appeared, from the bottom to the top of the film.
Automating DNA sequencing involves automating the process of detecting the fragments on the electrophoresis gel and then automatically determining the DNA base sequence from the sequence of detected fragments using the above algorithm implemented in a microprocessor. Because of the time needed to expose the x-ray film to the beta radiation of the P-32 phosphorus, and other considerations involving the use of radioisotopes, new methods of tagging and sequencing based on fluorescence were developed. See
Biophysical and Biochemical Aspects of Fluoresene Spectroscopy
, edited by T. Gregory Dewey, Plenum Press, 1997; “Large Scale and Automated Sequence Determination,” by T. Hunkspillar, et al.,
Science
, Vol. 254, pages 59-67 (1991) and “DNA Sequencing: Present Limitations and Prospects for the Future,” by B. Barrell, the FASEB Journal, Vol. 5, page 40-45 (1991).
Fluorescence tagging of the fragments involves the attachment of a fluorescent compound, or fluorophore, to each fragment analogously to the attachment of the radioactive label to each fragment. These fluorescence labels were found to not adversely affect the process of gel electrophoreses or sequence.
Fluorescence is an optical method that involves stimulating the fluorescent molecule by shining light on it at an optical wavelength that is optimum for that molecule. Fluorescent light is then given off by the molecule at a characteristic wavelength that is typically slightly longer than the stimulation wavelength. By focusing the light at the stimulating wavelength down to a point on the gel and then detecting the presence of any optical radiation at the characteristic wavelength of light from the fluorescent molecule, the presence at that point of fragments of DNA tagged with that fluorescent molecule may be determined.
Two methods of implementing an automated DNA sequencing instrument are known in the art. One, reported by Smith et al., in
Nature
, Vol 321, pages 674-679 (1986), puts a different fluorescent tag on each of the four samples of fragments described above. Thus, the sample of fragments that end in the base A are tagged by one fluorophore; the sample of fragments that end in the base G are tagged by another fluorophore, and so on for the other two samples. Each fluorophore can be distinguished by its own stimulation and emission wavelengths of light.
In the Smith et al. method, all four samples are electrophoresed in the same lane together and the differences in their tags are used to distinguish them. That has the advantage that four separate lanes are not used, since the progression of fragments in different lanes is often not consistent with one another and difficulties often arise in determining the sequence as a result.
Another method, reported by Ansor et al., in
J. Biochem Biophys. Methods
, Vol. 13, pages 315-323 (1986) and
Nucleic Acids Res
., Vol 15(11), pages 4593-4602 (1987), uses one fluorescent tag for all fragments, but employs four separate lanes of gel electrophoresis in a manner that is similar to radioactive labeled sequencing. That approach has the potential disadvantage that four lanes, with different fragment migration rates caused by local temperature variations and other inconsistencies within the gel, could limit the reliability of the sequence determination.
Fluorescence tagging and the detection of natural fluorescence in molecules is a method of analytical chemistry and biology that is well known in the art. The methods described above have been developed for DNA sequencing by the creation of fluorescent tags that can be bound to fragments of DNA. The instruments used to detect fluorescence consist of the following parts. A light source with a broad optical bandwidth such as a light bulb or a laser is used as the source of the stimulating light. An optical filter is used to select the light at the desired stimulation wavelength and beam it onto the sample. Optical filters are available at essentially any wavelengt
Alexander Lyle A.
Blank Rome Comisky & McCauley LLP
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