Automatic sequencer/genotyper having extended spectral response

• US 5,871,628 A
• Filed: 08/22/1996
• Issued: 02/16/1999
• Est. Priority Date: 08/22/1996
• Status: Expired due to Fees

• Current Assignee: Science Applications International Corporation, Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Science Applications International Corporation, University of Texas System
• Inventors: Dabiri, Ali, Garner, Harold R.
• Primary Examiner(s): Beisner, William H.
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/702,767
• Time in Patent Office: 908 Days
• Field of Search: 204/603, 204/612, 204/452, 204/461
• US Class Current: 204/461
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: G01N 27/44721 by optical means

Abstract

An advanced imaging spectrograph system provides for slab-gel DNA sequencing and genotyping with high throughput sequencing. The system is based on the integration of improved electrophoresis structures with an imaging spectrophotometer that records the entire emission spectra along an imaging line across a sequencing gel (or capillary array). The system includes spectral shape matching to improve dye identification allowing the use of dyes having nearly any emission spectra and allowing greater than four dye multiplexing.

Claims

1. Apparatus for detecting charged molecules having differing mobilities and chemical properties and having fluorescent tags, each tag corresponding to a particular chemical property of the molecules, comprising:

a plurality of electrophoresis lanes, each lane having a respective first and second end, and an electrophoresis medium between the first and second end of each lane, wherein an electrical potential, of appropriate polarity, is applied between the first and second end causes the charged molecules, applied at the first end, to travel toward the second end at a rate proportional to the molecule'"'"'s mobility such that the charged molecules are separated along the lane based on the molecules mobility;

a read zone that extends substantially along an image line that intersects the plurality of electrophoresis lanes near the second ends;

a light source that illuminates the read zone with excitation light to cause the charged molecules to fluoresce producing fluorescent light; and

a hyperspectral imager that spectrally images the fluorescent light onto a two-dimensional imaging plane, the first dimension being associated with a distance along the image line of the read zone and the second dimension being associated with the wavelength of the fluorescent light, the hyperspectral imager having continuous channels less than about 5 nanometers wide and including means for providing a spectral resolution of about 3-4 nanometers, contiguously spaced between about 450 nanometers and 900 nanometers.

2. Apparatus for detecting fluorescent molecules as defined in claim 1, further comprising:

a camera having a two dimensional pixel array, the camera generating video signals based on the intensity of light incident upon the pixel array.

3. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein:

the hyperpsectral imager further comprises a linear entrance aperture with discrete locations along the aperture corresponding to locations along the first dimension of the image plane; and

the apparatus further comprises a plurality of optical fibers that couple the fluorescent light from the read zone to corresponding locations along the entrance aperture.

4. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the hyperspectral imager includes an optical lens system that directly images the read zone.

5. Apparatus for detecting fluorescent molecules as defined in claim 1, further comprising 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.

6. Apparatus for detecting fluorescent molecules as defined in claim 5, wherein:

the charged molecules are at least 10 differing genetic markers of a genome;

a fluorescent tag, having a unique spectral profile, is associated with each genetic marker; and

the processor compares the detected fluorescent light with the reference spectral profiles by calculating weighting factors f or each reference spectral profile based on a deconvolution of the detected fluorescent light with the reference spectral profile for each of the fluorescent tags.

7. Apparatus for detecting fluorescent molecules as defined in claim 5, wherein:

at least one fluorescent dye is attached to charged molecules of a known mobility, to provide a mobility calibration reference; and

the processor calculates the mobility of any charged molecules of unknown mobility, based on the unknown molecule'"'"'s travel rate and the mobility calibration reference.

8. Apparatus for detecting fluorescent molecules as defined in claim 5, wherein:

the fluorescent tags used in adjacent lanes differ from each other; and

the processor tracks lane drift along the image line using the difference between the fluorescent tags in adjacent lanes.

9. Apparatus for detecting fluorescent molecules as defined in claim 5 wherein at least one additional fluorescent tag is used in every other lane.

10. Apparatus for detecting fluorescent molecules as defined in claim 9 wherein four fluorescent tags are used in each lane to identify the bases C, G, A and T, respectively, of a DNA fragment, and wherein a fifth fluorescent tag is added to every second lane for use in lane tracking.

11. Apparatus for detecting fluorescent molecules as defined in claim 1, further comprising a display that displays a graph of the chemical property of the molecules crossing the imaging line verses time.

12. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the charged molecules are DNA fragments, each fragment being tagged with an attached fluorescent dye that identifies the fragment'"'"'s end base.

13. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the fluorescent tags are four primer dyes, FITC, TRITC, NBD-fluoride, and Texas Red, corresponding to the bases C, G, A, and T, respectively.

14. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the light source generates excitation light at wavelengths of about 488 nanometers and 514 nanometers.

15. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein:

the lanes are on a planar substrate;

the excitation light is a laser beam that travels along the image line to simultaneously illuminate the lanes.

16. Apparatus for detecting fluorescent molecules as defined in claim 15, further comprising a mirror that reflects the laser beam back through the read zone along the image line to increase the intensity and uniformity of the fluorescent light.

17. Apparatus for detecting fluorescent molecules as defined in claim 15, further comprising a lens that couples the laser beam from a laser to the gel to increase the intensity of the excitation light traveling along the image line.

18. Apparatus for detecting fluorescent molecules as defined in claim 1, further comprising:

optical fibers for coupling the fluorescent light from the image line to an entrance slit on the imaging spectrometer;

a cylindrical lens that focuses the fluorescent light from the imaging line onto ends of the optical fibers; and

a mirror located behind the read zone such that fluorescent light travelling away from the fiber end is reflected back toward the optical fiber ends.

19. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the second dimension of the imaging plane corresponds to the spectral range from about 450 nanometers to about 900 nanometers.

20. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein:

the excitation light is narrowband light having a wavelength within a spectral range of the imaging spectrometer such that excitation light scattered by the electrophoresis gel is imaged on a pixel array on the imaging plane;

the electrical signals, generated by the-pixel array based on the intensity of light collected by pixels of the pixel array corresponding to the wavelength of the excitation light, provide a monitor of the excitation light'"'"'s intensity.

21. Apparatus for detecting fluorescent molecules as defined in claim 1, further comprising a buffer to optically couple the light from the light source to electrophoresis medium.

22. Apparatus for detecting fluorescent molecules as defined in claim 1, further comprising first and second electrodes for applying the electrical potential to the lanes at the first and second ends, respectively, and extending across all of the plurality of lanes.

23. Apparatus for detecting fluorescent molecules as defined in claim 22, further comprising a plurality of loading electrodes situated near the first electrode, each loading-electrode associated with a lane for loading the charged molecules into the lanes.

24. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the electrophoresis medium is a gel having a thickness of 200 microns which is sandwiched between two glass plates.

25. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the read zone further includes fluorescent markers at each end of the image line for indicating the read zone.

26. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the plurality of electrophoresis lanes comprise 384 separate parallel lanes.

27. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the hyperspectral imager simultaneously images the fluorescent light onto the two-dimensional imaging plane without any scanning motions or delays.

28. Apparatus for detecting fluorescent molecules as defined in claim 1, wherein the plurality of electrophoresis lanes comprise a capillary electrophoresis array.

29. Apparatus for detecting fluorescent molecules as defined in claim 1, further comprising a heater that is thermally coupled to the electrophoresis medium for maintaining the medium within a predetermined temperature range.

30. A method for sequencing DNA, comprising:

producing DNA fragments that are tagged with fluorescent dyes, the dyes indicating an end base associated with the respective DNA fragment;

separating the DNA fragments according to mobility using electrophoresis of the fragments on a plurality of electrophoresis lanes, the separated DNA fragments forming fragment groups of slightly different mobility;

exciting the fragment groups with excitation light to cause the fluorescent tags to fluoresce;

forming a series of hyperspectral images of the separated DNA fragments, the hyperspectral images simultaneously covering all of the electrophoresis lanes and a broad spectral range; and

identifying the fluorescent dye associated with DNA fragments of particular molecular weight by fitting the spectra emitted by a fragment group with reference spectra associated with the fluorescent dyes.

31. Apparatus for detecting charged molecules having differing mobilities and chemical properties and having fluorescent tags, each tag corresponding to a particular chemical property of the molecules, comprising:

a plurality of electrophoresis lanes, each lane being on a planar substrate and having a respective first and second end, and an electrophoresis medium between the first and second end of each lane, wherein an electrical potential, of appropriate polarity, is applied between the first and second end causes the charged molecules, applied at the first end, to travel toward the second end at a rate proportional to the molecule'"'"'s mobility such that the charged molecules are separated along the lane based on the molecule'"'"'s mobility;

a read zone that extends substantially along an image line that intersects the plurality of electrophoresis lanes near the second ends;

a laser source that illuminates the read zone with a laser beam along the image line to simultaneously illuminate the lanes, causing the charged molecules to fluoresce producing fluorescent light;

a mirror that reflects the laser beam back through the read zone along the image line to increase the intensity and uniformity of the flourescent light; and

an imaging spectrometer that spectrally images the fluorescent light onto a two-dimensional imaging plane, the first dimension being associated with a distance along the image line of the read zone and the second dimension being associated with the wavelength of the fluorescent light.

32. Apparatus for detecting charged molecules having differing mobilities and chemical properties and having fluorescent tags, each tag corresponding to a particular chemical property of the molecules, comprising:

a plurality of electrophoresis lanes, each lane having a respective first and second end, and an electrophoresis medium between the first and second end of each lane, wherein an electrical potential, of appropriate polarity, is applied between the first and second end causes the charged molecules, applied at the first end, to travel toward the second end at a rate proportional to the molecule'"'"'s mobility such that the charged molecules are separated along the lane based on the molecule'"'"'s mobility;

a read zone that extends substantially along an image line that intersects the plurality of electrophoresis lanes near the second ends;

a light source that illuminates the read zone with excitation light to cause the charged molecules to fluoresce producing fluorescent light;

an imaging spectrometer that spectrally images the fluorescent light onto a two-dimensional imaging plane, the first dimension being associated with a distance along the image line of the read zone and the second dimension being associated with the wavelength of the fluorescent light;

optical fibers for coupling the fluorescent light from the image line to an entrance slit on the imaging spectrometer;

a cylindrical lens that focuses the fluorescent light from the imaging line onto ends of the optical fibers; and

a mirror located behind the read zone such that fluorescent light travelling away from the fiber end is reflected back toward the optical fiber ends.

33. Apparatus for detecting charged molecules having differing mobilities and chemical properties and having fluorescent tags, each tag corresponding to a particular chemical property of the molecules, comprising:

a plurality of electrophoresis lanes, each lane having a respective first and second end, and an electrophoresis medium between the first and second end of each lane, and further comprising first and second electrodes situated to apply an electrical potential to the lanes at the first and second ends, respectively, wherein an electrical potential, of appropriate polarity, applied between the first and second electrodes causes the charged molecules, applied at the first end, to travel toward the second end at a rate proportional to the molecule'"'"'s mobility such that the charged molecules are separated along the lane based on the molecule'"'"'s mobility;

a plurality of loading electrodes situated near the first electrode, each loading electrode being associated with a lane for loading the charged molecules into the lanes;

a read zone that extends substantially along an image line that intersects the plurality of electrophoresis lanes near the second ends;

a light source that illuminates the read zone with excitation light to cause the charged molecules to fluoresce producing fluorescent light; and

an imaging spectrometer that spectrally images the fluorescent light onto a two-dimensional imaging plane, the first dimension being associated with a distance along the image line of the read zone and the second dimension being associated with the wavelength of the fluorescent light.

34. A method for detecting and sequencing charged molecules having differing mobilities and chemical properties, the charged molecules being fragments of a larger molecule and the detection thereof permitting sequencing of the larger molecule, the method comprising:

tagging the respective fragments with flourescent dyes, the dyes indicating an end base associated with the respective fragment;

separating the fragments according to mobility using electrophoresis of the fragments on a plurality of electrophoresis lanes, the separated fragments forming fragment groups of slightly different mobility;

illuminating a read zone associated with each electrophoresis lane with an excitation light source as the fragments pass therethrough, the excitation light source causing the charged molecules to fluoresce producing fluorescent light; and

hyperspectrally imaging the fluoresced light onto a series of two-dimensional planes, the hyperspectral images thus formed simultaneously covering all of the electrophoresis lanes and a broad spectral range.

35. The method of claim 34 wherein the step of hyperspectrally imaging comprises forming a hyperspectral image having a first dimension associated with a distance along an image line of the read zone and a second dimension associated with the wavelength of the fluoresced light, and further having continuous channels less than about 5 nanometers wide and having a spectral resolution of about 3-4 nanometers, continuously spaced between about 450 nanometers and 900 nanometers.

36. The method of claim 34 wherein the charged molecules which are grouped into respective fragments comprise DNA fragments.

37. The method of claim 34 further including identifying the fluoresced light with a dye associated with DNA fragments of particular molecular weight by fitting the spectra emitted by a fragment group with reference spectra associated with fluoresced light from a reference dye.