Image analysis – Applications – Biomedical applications
Reexamination Certificate
1997-05-18
2001-02-27
Mehta, Bhavesh (Department: 2721)
Image analysis
Applications
Biomedical applications
C382S190000
Reexamination Certificate
active
06195449
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field
The present invention relates generally to the field of nucleotide investigations, and more particularly to the detection and analysis of emission spectra generated during observation of excited fluorphore labelled nucleotide polymers undergoing separation by size, such as is done during the sequencing of bases in nucleotide polymers.
2. State of the Art
The genetic material of higher organisms comprises two strands of DNA. Each DNA strand is a polymer of nucleotide monomers and each monomer consists of a sugar residue (deoxyribose), a phosphate residue, and a purine or pyrimidine base. The monomers are linked in a continuous chain by a phosphoribosyl backbone. The double stranded DNA prefers a helical orientation and exists as a long linear strand in higher organisms (up to several centimeters in length in man) with its phosphoribosyl backbone oriented outwardly of the helix and the sequentially ordered bases oriented inwardly along the axis of the helix whereby complementary hydrogen bonding between bases hold the two strands together. By complementary it is understood that adenine nearly always forms hydrogen bonds with thymine and cytidine with guanidine. The phosphoribosyl backbone has a free hydroxyl group at the 3′ position extending from the terminal deoxyribose residue at one end and a free terminal phosphate group attached at the 5′ position of its last deoxyribose residue at the other, thus giving a directional orientation to the opposing strands.
It is the sequence of the four bases found on the strands of DNA, (denoted A, G, T, and C), that is the genetic code directing the synthesis of all the polypeptides or proteins (enzymes, collagen, muscle, etc. synthesized as a linear sequence of amino acid monomers). These polypeptides perform the metabolic processes essential to life and health and provide structure and mobility to organisms. The code is based on a sequence of three bases, thus 4
3
or 64 “code words” exist in the code. One triplet code is a start command, directing the initiation of synthesis of amino acid polymers (polypeptides), most triplets code for a particular amino acid to be added to the linear polypeptide chain, and a few triplet codes are stop commands directing the termination of synthesis of the polypeptide. A gene consists of the series of triplet codes, that is, the DNA sequence which directs the synthesis of a single protein. One gene codes for one protein. The industrial and research community desire to learn the sequence of DNA in all genes in humans and some other organisms and thereby harness this genetic code for a variety of useful purposes. With over 3×10
9
bases making up the genes in humans, the enormity of the task of determining their sequence as they occur in the genes is readily appreciated.
While the above discussion has used the term DNA and referred to DNA sequencing, and uses the terms DNA and DNA sequencing hereinbelow, it is understood that the invention has application to sequencing methods of any nucleotide polymer, e.g., amplified microsatellite nucleotide polymers and other methods involving the use of fluorescently tagged nucleotide polymer fragments used to generate a chromatogram.
Two methods of sequencing form the basis for large scale sequencing operations, a so-called chemical method and an enzymatic method. The enzymatic method exploits the process of DNA replication which always occurs in the 5′ to 3′ direction by the addition of a new nucleotide to the 3′ terminus of the growing DNA polymer catalyzed by an enzyme, DNA polymerase. The process is known as primer extension and the method of sequencing upon which it is based is the enzymatic or dideoxy method of DNA sequencing. Sanger et al.,
Proc. Natl. Acad. Sci.,
U.S.A. 74, 5463-5467 (1977). The chemical method of DNA sequencing was developed by A. M. Maxam and W. Gilbert, and is described in
Proc. Natl. Acad. Sci.,
Vol. 74, p. 560 (1977). Each method is well known and well described in the references cited above and are equally applicable to the invention. Suffice it to say, they involve a number of steps and result in fragments of DNA of varying sizes that end with a different base (A, T, C, or G). The determination of DNA sequence in these methods depends on separating the DNA fragments produced by order of size and either by what base they contain (when each lane has only one reaction product) or by what fluorophore tag is detected if all four reaction products are in one lane as in commercially popular sequencing machines. If the shortest fragment ends in A, then the first base in the sequence is A. If the next longest fragment ends in T, then the next base in the DNA sequence is T and so on. This is the basic algorithm for “base calling”, i.e., determining the sequence of purine and pyrimidine bases in a strand of DNA.
One commercially popular automatic sequencer, the ABI 373A®, available from Applied Biosystems, Inc., Foster City, Calif., performs the following steps after a nucleotide polymer is sampled and reaction products of varying length obtained. The reaction product fragments are tagged with a fluorophore, resolved by size by inducing them to migrate through a polyacrylamide gel via an electrical charge across the gel (gel-electrophoresis), exposed to an electromagnetic wave source to induce the emission of electromagnetic energy (fluorescence by the tag), and the emitted energy detected by a detector to produce an analog signal. The analog signal is sampled and the sampled values transmitted to a data file referred to as a gel file. The gel file data is then “tracked” and processed by ABI Sequencer® analyzer software which generates chromatogram data and stores it in a chromatogram data file. The software then automatically determines the DNA base sequence from the chromatogram data and stores the sequence data as part of the chromatogram data file. Examples of patented automatic sequencing apparatus and methods include U.S. Pat. No. 4,811,218 to Hunkapiller et al. issued Mar. 7, 1989 assigned to Applied Biosystems, Inc. (ABI), and U.S. Pat. No. 5,556,790 to Pettit issued Sep. 17, 1996, the disclosures of which are incorporated herein by reference. These methods and such commercially available instruments as the ABI 373A® as discussed and the Pharmacia A.L.F.®, from Pharmacia, Inc. of Piscataway, N.J., and the Licor® Sequencer from Licor of Lincoln, Nebr. all produce a chromatogram data file from an analog signal in a manner compatible with the initial steps of the present invention. It is understood that should newer methods of creating chromatogram data files be produced, they too would be compatible with the invention. In addition to the instruments and methods discussed above, other methods employing capillary electrophoresis can be used to produce a data file compatible with the initial steps of the invention. The initial steps of producing reaction products is the same, however, a gel is not used during the fragment separation step and, at least in one commercially popular machine, a CCD camera is used to detect fluorophore emission spectra. Other prior art methods not necessarily directed to gene sequencing, such as microsatellite amplification, employ fluorophore labeled nucleotides and generate signals that can be converted into chromatogram data files as well. These and yet to be developed methods which produce a signal that can be converted into a digital data file such as a chromatogram data file are compatible with the initial steps of the invention.
Some current commercial automated sequencers utilize a single gel plate which can accommodate up to 64 migration lanes simultaneously, that is, 64 unique DNA samples. The multiple lanes are generally run through the detector and detected simultaneously to increase the throughput of the sequencer. A single run on such a gel can result in collection of between 4000 and 9000 data points (one each 6 seconds) for each sample by means of intermittent sampling of the raw data generated by the detector for a gel pl
Bogden Robert
Leeuw Marcel De
Mallinckrodt Robert R.
Mallinckrodt & Mallinckrodt
Mehta Bhavesh
Patel Kanji
Peer Peter M.
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