Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
1999-01-08
2002-07-30
Hoff, Marc S. (Department: 2857)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C702S027000, C702S028000
Reexamination Certificate
active
06427126
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to DNA sequencing and genotyping, and more particularly, to DNA sequencers and genotypers that use optical fluorescence detection techniques.
The basic biological characteristics of a living organism are contained in its genes or genetic code. In humans, for example, a person's biological characteristics are controlled by the genetic code contained in 23 chromosome pairs. Each chromosome contains differing genes.
The specific details of a genetic code are contained in long double helical molecules called deoxyribonucleic acid or DNA. The DNA consists of long sequence pairs of four nucleotides or bases: adenosine, cytosine, guanosine or thymidine, commonly referred to by the letters A, C, G, and T, respectively. In the double helix, the A and T nucleotides are complementary and the C and G nucleotides are complementary. Thus, the DNA molecules consist of two complementary strands that are bound together by the complements.
It is often advantageous to know the sequence of the DNA nucleotides associated with a particular gene. For example, genetic defects can be detected by analyzing an organism's genes. The DNA nucleotides for several bacteria and viruses have been sequenced, and currently, sequencing of the entire human genome is in progress. The entire human DNA consists of approximately 3 billion nucleotides or base pairs.
Existing high speed DNA sequencers use electrophoresis gel techniques, in conjunction with fractioning enzymes and fluorescent tags or markers, to separate residual DNA sequence fragments as they travel through a gel. More specifically, each DNA fragment has an incrementally different molecular weight and size. Because the mobility of these DNA fragments through a gel is related to the fragment's weight, structure, and charge, the differing fragments travel through the gel at differing speeds. Thus, the time it takes a fragment to travel through the gel, i.e., it's mobility, relates to the fragment's size and charge.
Generally, four fluorescent tags are used to visualize DNA fragments. These tags bind on the residual fragments in accordance with the exposed end base, if using dye terminator chemistry, or are attached to primers that are used to initiate the sequencing reaction, if using dye primer chemistry. The sequence is read by causing the fluorescent markers to fluoresce. The four fluorescent tags generally are selected to have a strong fluorescence peak that is separated from the strong fluorescence peak of the remaining tags. An optical instrument detects the emitted fluorescence signals.
Existing DNA Sequencers use an optical filter having a pass-band that is centered about the appropriate wavelength to distinguish between the dyes, and thus the fragments. The optical instrument typically includes a simple spectrometer or a filter wheel and a photomultiplier. The filter wheel has several colored filters, each filter passing light within a wavelength band corresponding to the spectral peak of one of the tags. A simple spectrograph has a wavelength-dependent light disperser such as a prism. The light disperser spreads, generally along a line, the different wavelengths of fluorescent light from the DNA fragments traveling in the gel. Four detectors are placed along the spreading line of the spectrograph at differing locations that correspond to the wavelengths associated with the fluorescent tags.
Fluorescent dyes have been found to be good fluorescent tags. Thus, for example, using dye primer chemistry, the tag usually used in association with the C base is fluorescein-5-isothiocynate (FITC), which has an emission or fluorescence peak at about 525 nanometers. The tag often associated with the T base is Texas Red, which has a fluorescence peak at about 620 nanometers. The tag often associated with the G base is Tetramethyl rhodamine isothiocynate (TRITC), which has a fluorescence peak at about 580 nanometers, and the marker usually associated with the A base is 4-fluoro-7nitro-benzofurazan (NBD-fluoride), which has a fluorescence peak at about 540 nanometers. Commercially, four universal primers, respectively labeled with dyes called FAN (C), TAMRA (G), JOE (A), and ROX (T), are available from Applied Biosystems, Inc. (ABI) of Foster City, Calif.
The fluorescent dyes indicated above are subject to bleaching which limits the excitation light's power level and thus the intensity of the emitted fluorescence signal from the dyes. The upper limits on fluorescence intensity limit the signal-to-noise ratio (SNR) and eventually the system's throughput.
Accordingly, there exists a need for a sequencing or genotyping system that has increased throughput and sensitivity over systems using four dyes that are distinguished by their respective fluoresce peaks. The present invention satisfies these needs.
SUMMARY OF THE INVENTION
A sequencing or genotyping system according to the present invention includes an imaging spectrograph that records the entire emission spectra across a plurality of lanes in an electrophoresis sequencing gel. The system includes spectral shape matching to improve dye identification, thereby allowing the use of dyes having nearly any emission spectra and allowing greater than four dye multiplexing.
In a first embodiment of the invention, the system includes a plurality of electrophoresis lanes. Each lane has a respective first and second end, and an electrophoresis medium between the first and second ends. Each lane is loaded with fluorescently-tagged charged molecules having differing mobilities and chemical properties. An electrical potential, of appropriate polarity, is applied between the first and second ends and causes charged molecules applied at the first end to travel toward the second end at a rate proportional to each molecule's mobility such that the charged molecules are separated along the lane based on the molecule's mobility. A “read zone” extends substantially along an image line and intersects the plurality of electrophoresis lanes near the second ends. The system also includes a light source and an imaging spectrometer. The light source illuminates the read zone with excitation light to cause the charged molecules to fluoresce and produce fluorescent light. The imaging spectrometer spectrally images the fluorescent light onto a two-dimensional imaging plane. The first dimension of the imaging plane is associated with a distance along the image line of the read zone and the second dimension is associated with the wavelength of the fluorescent light. The imaging spectrometer simultaneously images the fluorescent light onto the two-dimensional imaging plane without any scanning motions or delays.
The system may further include a camera having a two dimensional pixel array. The camera generates video signals based on the intensity of light incident upon the pixel array. A display may display a graph of the chemical properties of the molecules crossing the imaging line verses time.
In more detailed features of the invention, the imaging spectrograph further comprises a linear entrance aperture with discrete locations along the aperture corresponding to locations along the first dimension of the image plane. Further, a plurality of optical fibers couple the fluorescent light from the read zone to corresponding locations along the entrance aperture. Alternatively, the imaging spectrograph may include an optical lens system that directly images the read zone. Further, the system may include a processor that compares the detected fluorescent light received from molecules of a given mobility with reference spectral profiles for the fluorescent tags to identify the associated fluorescent tag and thus the molecule's associated chemical property.
In other more detailed features of the invention, associated with genotyping or DNA fingerprinting, the charged molecules include at least 10 different genetic markers of a genome, and a fluorescent tag, having a unique spectral profile, is associated with each genetic marker. The proces
Dabiri Ali
Garner Harold R.
Barbee Manuel L.
Fitch Even Tabin & Flannery
Hoff Marc S.
Science Applications International Corporation
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