AUTOMATED HYBRIDIZATION/IMAGING DEVICE FOR FLUORESCENT MULTIPLEX DNA SEQUENCING

• US 20020012910A1
• Filed: 11/28/1995
• Published: 01/31/2002
• Est. Priority Date: 10/22/1993
• Status: Abandoned Application

• Current Assignee: Alvin W. Kimball, Diane M. Dunn, F. Mark Ferguson, Joshua L. Cherry, Leonard J. Di Sera, Raymond F. Gesteland, Robert B. Weiss
• Original Assignee: Alvin W. Kimball, Diane M. Dunn, F. Mark Ferguson, Joshua L. Cherry, Leonard J. Di Sera, Raymond F. Gesteland, Robert B. Weiss
• Inventors: FERGUSON, F. MARK, GESTELAND, RAYMOND F., DUNN, DIANE M., WEISS, ROBERT B., CHERRY, JOSHUA L., KIMBALL, ALVIN W., DI SERA, LEONARD J.
• Application Number:
  US08/563,462
• Publication Number:
  US 20020012910A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/44721 by optical means

Abstract

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

Claims

1. A method for sequencing a nucleic acid specimen by automated multiplex sequencing comprising the steps of:

(a) preparing multiplex sequencing reaction products including at least one tag sequence in a plurality of vessels, wherein said products in each vessel differ in length from each other and all terminate at a fixed known nucleotide or nucleotides, wherein said fixed known nucleotide or nucleotides in one of said vessels differ from said fixed known nucleotide or nucleotides in another of said vessels;

(b) fractionating said products from each said vessel according to their size;

(c) attaching said fractionated products from each said vessel to a membrane;

(d) placing said membrane containing said fractionated products in a chamber device of an integrated automated imaging hybridization chamber system comprising an hybridization chamber device, means for fluid delivery to said chamber device, imaging means for light delivery to said membrane and image recording of fluorescence emanating from said membrane while in said chamber device, and controller means for controlling operation of said system;

(e) introducing a first oligonucleotide probe containing a binding moiety to which an enzyme may be bound and which is able to hybridize specifically with one of said tag sequences into said chamber device by said fluid delivery means and bringing said probe into contact with said membrane thereby hybridizing said probe to said fractionated products;

(f) introducing an enzyme into said chamber device by said fluid delivery means and bringing said enzyme into contact with said first oligonucleotide probe thereby binding the enzyme to said binding moiety on said first oligonucleotide probe;

(g) introducing a fluorogenic substrate into said chamber device by said fluid delivery means and bringing said substrate into contact with said enzyme, wherein said enzyme converts said fluorogenic substrate into a fluorescent product and wherein the fluorescent product interacts with the membrane and is spatially localized thereon;

(h) illuminating said fluorescent product in said chamber device with a beam of light from said imaging means capable of exciting fluorescence by said fluorescent product, wherein said fluorescence produces a pattern of hybridization that reflects the nucleotide sequence of said nucleic acid specimen;

(i) imaging said pattern of hybridization by said imaging means, wherein said pattern of hybridization is stored as a series of digital signals; and

(j) converting said series of digital signals by said controller means into a linear string of nucleotides corresponding to the nucleotide sequence of the nucleic acid specimen.

2. The method of claim 1 further comprising

(k) removing the fluorescent product from the membrane by introducing an appropriate wash solution into the chamber compartment by the fluid delivery means and removing the wash solution from the chamber compartment by means for fluid removal from said chamber compartment via valve means contained in the lower portion of said outer cylinder sidewall;

(l) introducing a second oligonucleotide probe, containing a binding moiety to which an enzyme may be bound and which is able to hybridize specifically with one of said tag sequences different than the tag sequence of step (e), into said chamber device by said fluid delivery means and bringing said second oligonucleotide probe into contact with said membrane, thereby hybridizing said second probe to said fractionated products;

(m) introducing an enzyme into said chamber device by said fluid delivery means and bringing said enzyme into contact with said second oligonucleotide probe, thereby binding the enzyme to said binding moiety on said second oligonucleotide probe; and

(n) repeating steps (g) through (j).

3. The method of claim 1 wherein step (b) further comprises fractionating said products by gel electrophoresis.

4. The method of claim 3 wherein step (c) further comprises transferring said fractionated products to said membrane by electrophoresis.

5. The method of claim 4 wherein said electrophoresis is direct transfer electrophoresis.

6. The method of claim 1 wherein the hybridization chamber device used in step (d) comprises

generally horizontal, concentrically nested inner and outer cylinders having a common axis,

the inner cylinder consisting of a cylindrical sidewall having an exterior surface and sealed ends, said inner cylinder being rotatable about said axis,

the outer cylinder consisting of a stationary cylindrical sidewall having interior and exterior surfaces, a transparent window contained therein and having sealable ends,

said inner cylinder being rotatable within said outer cylinder about said axis, said membrane being mounted on the exterior sidewall surface of said inner cylinder;

said inner and outer cylinders having diameters such that there is a space between the respective sidewalls of said inner and outer cylinders defining a chamber compartment of sufficient width that said inner cylinder can rotate within said outer cylinder without said membrane coming in contact with the interior surface of said outer cylinder sidewall,

said chamber compartment being in fluid communication with said means for fluid delivery via valve means contained in a lower portion of said outer cylinder sidewall.

7. The method of claim 6 wherein said hybridization chamber device further comprises means for rotating said inner cylinder.

8. The method of claim 7 wherein said rotating means comprises a stepper motor.

9. The method of claim 6 wherein said fluid delivery means comprises a plurality of reservoirs for storing fluids and metering pumps for delivering fluid in selectable volumes.

10. The method of claim 9 wherein said fluid delivery means further comprises a batch heater for heating said fluids to a selected temperature before said fluids are delivered to said chamber compartment.

11. The method of claim 9 wherein said fluid delivery means further comprises a probe concentrate reservoir for storing a concentrated liquid hybridization probe and the like, and means for mixing said concentrated hybridization probe and a fluid to form a hybridization solution and delivering said hybridization solution to said chamber compartment.

12. The method of claim 11 wherein said fluid delivery means further comprises means for chilling said hybridization probe.

13. The method of claim 6 wherein said hybridization chamber device further comprises aheating element for heating a fluid contained in said chamber compartment to a selected temperature.

14. The method of claim 13 wherein said hybridization chamber device further comprises a thermocouple for monitoring the temperature of a fluid contained in said chamber compartment.

15. The method of claim 6 further comprising means for fluid removal from said chamber compartment via valve means contained in the lower portion of said outer cylinder sidewall.

16. The method of claim 6 wherein said imaging means includes a light source for producing and delivering a beam of light of an appropriate wavelength to the membrane for exciting fluorescence of said fluorescent product.

17. The method of claim 16 wherein said light source comprises a laser.

18. The method of claim 16 wherein said light source comprises a tungsten-halogen lamp with a band pass filter.

19. The method of claim 16 wherein said imaging means further comprises a filter for removing undesirable wavelengths of light from said beam of light.

20. The method of claim 16 wherein said imaging means includes a CCD camera for recording an image of said fluorescence emanating from said fluorescent product on said membrane and a filter for removing interfering wavelengths of light from said fluorescence of said fluorescent product.

21. The method of claim 20 wherein said CCD camera is operable in Time Delay and Integration mode.

22. The method of claim 1 wherein step (f) further comprises attaching to said enzyme a molecule having an affinity for binding to said binding moiety, said molecule forming a bridge for binding of said enzyme to said binding moiety.

23. The method of claim 22 wherein said binding moiety comprises a biotin molecule covalently attached to said probe and said bridging molecule is selected from the group consisting of streptavidin and avidin.

24. The method of claim 22 wherein said binding moiety comprises a digoxigenin molecule covalently attached to said probe and said bridging molecule comprises an anti-digoxigenin antibody.

25. The method of claim 1 wherein the enzyme of step (f) is alkaline phosphatase.

26. The method of claim 1 wherein the fluorogenic substrate of step (g) is a phosphorylated benzothiazole derivative.

27. The method of claim 26 wherein the fluorogenic substrate is BBTP.

28. The method of claim 1 wherein step (i) further comprises scanning the full length of the membrane.

29. The method of claim 28 wherein said membrane is moved in synchrony with the scanning of the membrane, said movement actuated by the stepper motor coupled to the inner cylinder of the hybridization chamber device and controlled by the controller means.

30. The method of claim 1 wherein step (j) comprises converting said digital signals into a linear string of nucleotides corresponding to the nucleotide sequence of the nucleic acid specimen by an automated sequence reader.

31. An integrated automated imaging hybridization chamber system for sequencing a nucleic acid specimen by automated multiplex sequencing comprising an hybridization chamber device for mounting a membrane containing fractionated multiplex sequencing reaction products, means for fluid delivery to said chamber device, imaging means for light delivery to said membrane and image recording of fluorescence emanating from said membrane while in said chamber device, and controller means for controlling operation of the system.

32. The system of claim 31 wherein the hybridization chamber device comprises

generally horizontal, concentrically nested inner and outer cylinders having a common axis,

the inner cylinder consisting of a cylindrical sidewall having an exterior surface and sealed ends, said inner cylinder being rotatable about said axis,

the outer cylinder consisting of a stationary cylindrical sidewall having interior and exterior surfaces, a transparent window contained therein and having sealable ends,

said inner cylinder being rotatable within said outer cylinder about said axis, said membrane being mounted on the exterior sidewall surface of said inner cylinder;

said inner and outer cylinders having diameters such that there is a space between the respective sidewalls of said inner and outer cylinders defining a chamber compartment of sufficient width that said inner cylinder can rotate within said outer cylinder without said membrane coming in contact with the interior surface of said outer cylinder sidewall, said chamber compartment being in fluid communication with

said means for fluid delivery via valve means contained in the lower portion of said outer cylinder sidewall.

33. The system of claim 32 wherein said hybridization chamber device further comprises means for rotating said inner cylinder.

34. The system of claim 33 wherein said rotating means comprises a stepper motor.

35. The system of claim 32 wherein said fluid delivery means comprises a plurality of reservoirs for storing fluids and metering pumps for delivering fluid in selectable volumes.

36. The system of claim 35 wherein said fluid delivery means further comprises a batch heater for heating said fluids to a selected temperature before said fluids are delivered to said chamber compartment.

37. The system of claim 35 wherein said fluid delivery means further comprises a probe concentrate reservoir for storing a concentrated liquid hybridization probe and the like, and means for mixing said concentrated hybridization probe and a fluid to form a hybridization solution and delivering said hybridization solution to said chamber compartment in a selectable volume.

38. The system of claim 37 wherein said fluid delivery means further comprises means for chilling said hybridization probe.

39. The system of claim 32 wherein said hybridization chamber device further comprises an adhesive film heating element for heating a fluid contained in said chamber compartment to a selected temperature, wherein said heating element is coupled to the exterior surface of the sidewall of the outer cylinder.

40. The system of claim 39 wherein said hybridization chamber device further comprises a thermocouple for monitoring the temperature of fluid contained in said chamber compartment.

41. The system of claim 32 further comprising means for fluid removal from said chamber compartment via valve means contained in the lower portion of said outer cylinder sidewall.

42. The system of claim 32 wherein said imaging means includes a light source for producing and delivering a beam of light of an appropriate wavelength to the membrane for exciting fluorescence of said fluorescent product.

43. The system of claim 42 wherein said light source is selected from the group consisting of a laser and a tungsten-halogen lamp with a band pass filter.

44. The system of claim 43 wherein said imaging means further comprises a filter for removing undesirable wavelengths of light from said beam of light.

45. The system of claim 42 wherein said imaging means includes a CCD camera for recording an image of said fluorescence emanating from said fluorescent product on said membrane and a filter for removing interfering wavelengths of light from said fluorescence of said fluorescent product.

46. The system of claim 45 wherein said CCD camera is operable in Time Delay and Integration mode.

47. The system of claim 31 wherein the controller means is a programmable computer.

48. A method for enzyme-linked fluorescent detection of nucleic acid comprising the steps of:

(a) attaching the nucleic acid to be detected to a membrane;

(b) contacting the membrane with an oligonucleotide probe containing a binding moiety for binding an enzyme and which is able to hybridize specifically with a complementary sequence on the nucleic acid, thereby hybridizing said probe to said nucleic acid;

(c) contacting the probe with an enzyme thereby binding the enzyme to said binding moiety on the probe;

(d) contacting the enzyme with a fluorogenic substrate so that the enzyme converts the fluorogenic substrate into a fluorescent product, wherein the fluorescent product interacts with the membrane and is spatially localized thereon; and

(e) illuminating said fluorescent product with a beam of light capable of exciting fluorescence by said fluorescent product, wherein said fluorescence produces a pattern of hybridization that reflects the location of said nucleic acid on said membrane.

49. The method of claim 48 wherein said membrane is comprised of nylon.

50. The method of claim 48 wherein step (c) further comprises attaching to said enzyme a molecule having an affinity for binding to said binding moiety, said molecule forming a bridge for binding of said enzyme to said binding moiety.

51. The method of claim 50 wherein said binding moiety comprises a biotin molecule covalently attached to said probe and said bridging molecule is selected from the group consisting of streptavidin and avidin.

52. The method of claim 50 wherein said binding moiety comprises a digoxigenin molecule covalently attached to said probe and said bridging molecule comprises an anti-digoxigenin antibody.

53. The method of claim 50 wherein the enzyme is alkaline phosphatase.

54. The method of claim 53 wherein the fluorogenic substrate is a phosphorylated benzothiazole derivative.

55. The method of claim 54 wherein the fluorogenic substrate is BBTP.

56. The method of claim 50 further comprising

(f) imaging said pattern of hybridization by imaging means.

57. The method of claim 56 wherein said imaging means includes a light source for producing and delivering a beam of light of an appropriate wavelength to the membrane for exciting fluorescence of said fluorescent product.

58. The method of claim 57 wherein said light source is selected from the group consisting of a laser and a tungsten-halogen lamp with a band pass filter.

59. The method of claim 57 wherein said imaging means further comprises a filter for removing undesirable wavelengths of light from said beam of light.

60. The method of claim 57 wherein said imaging means includes a CCD camera for recording an image of said fluorescence emanating from said fluorescent product on said membrane and a filter for removing interfering wavelengths of light from said fluorescence of said fluorescent product.

61. The method of claim 60 wherein said CCD camera is operable in Time Delay and Integration mode.

62. A hybridization chamber device for hybridizing a probe to a nucleic acid bound to a membrane comprising

generally horizontal, concentrically nested inner and outer cylinders having a common axis,

the inner cylinder consisting of a cylindrical sidewall having an exterior surface and sealed ends, said inner cylinder being rotatable about said axis,

the outer cylinder consisting of a stationary cylindrical sidewall having interior and exterior surfaces,

a transparent window contained therein and having sealable ends,

A said inner cylinder being rotatable within said outer cylinder about said axis, said membrane being mounted on the exterior sidewall surface of said inner cylinder;

said inner and outer cylinders having diameters such that a there is a space between the respective sidewalls of said inner and outer cylinders defining a chamber compartment of sufficient width that said inner cylinder can rotate within said outer cylinder without said membrane coming in contact with the interior surface of said outer cylinder sidewall.

63. The hybridization chamber device of claim 62 wherein said hybridization chamber device further comprises means for rotating said inner cylinder.

64. The hybridization chamber device of claim 63 wherein said rotating means comprises a stepper motor.

65. The hybridization chamber device of claim 62 wherein said hybridization chamber device further comprises a heating element for heating a fluid contained in said chamber compartment to a selected temperature.

66. The hybridization chamber device of claim 69 wherein said hybridization chamber device further comprises a thermocouple for monitoring the temperature of a fluid contained in said chamber compartment.

67. The hybridization chamber device of claim 65 wherein said heating element is a film contained on the exterior surface of the said wall of the outer cyliner.

68. The hybridization chamber device of claim 62 further comprising means for fluid delivery, said chamber compartment being in fluid communication with said means for fluid delivery via valve means contained in a lower portion of said outer cylinder sidewall, wherein said fluid delivery means comprises a plurality of reservoirs for storing fluids and metering pumps for delivering fluid in selectable volumes.

69. The hybridization chamber device of claim 68 wherein said fluid delivery means further comprises a batch heater for heating said fluids to a selected temperature before said fluids are delivered to said chamber compartment.

70. The hybridization chamber device of claim 68 wherein said fluid delivery means further comprises a probe concentrate reservoir for storing a concentrated liquid hybridization probe and the like, and means for mixing said concentrated hybridization probe and a fluid to form a hybridization solution and delivering said hybridization solution to said chamber compartment.

71. The hybridization chamber device of claim 70 wherein said fluid delivery means further comprises means for chilling said hybridization probe.

72. The hybridization chamber device of claim 62 further comprising means for fluid removal from said chamber compartment via valve means contained in the lower portion of said outer cylinder sidewall.

73. The hybridization chamber device of claim 62 wherein said hybridization chamber device further comprises imaging means for light delivery to said membrane and image recording of fluorescence emanating from said membrane while in said hybridization chamber device, wherein said imaging means includes a light source for producing and delivering a beam of light of an appropriate wavelength to the membrane for exciting fluorescence of said fluorescent product.

74. The hybridization chamber device of claim 73 wherein said light source is selected from the group consisting of laser and a tungsten-halogen lamp with a band pass filter.

75. The hybridization chamber device of claim 74 wherein said imaging means further comprises a filter for removing undesirable wavelengths of light from said beam of light.

76. The hybridization chamber device of claim 73 wherein said imaging means includes a CCD camera for recording an image of said fluorescence emanating from said fluorescent product on said membrane and a filter for removing interfering wavelengths of light from said fluorescence of said fluorescent product.

77. The hybridization chamber device of claim 76 wherein said CCD camera is operable in Time Delay and Integration mode.

78. A method for detecting a macromolecule bound to a membrane comprising the steps of:

(a) preparing a sample of the macromolecule and attaching the macromolecule to a membrane;

(b) placing said membrane containing said macromolecule in a chamber device of an integrated automated imaging hybridization chamber system comprising an hybridization chamber device, means for fluid delivery to said chamber device, imaging means for light delivery to said membrane and image recording of fluorescence emanating from said membrane while in said chamber device, and controller means for controlling operation of said system;

(c) introducing a probe containing a binding moiety to which an enzyme may be bound and which is able to bind specifically with said macromolecule into said chamber device by said fluid delivery means and bringing said probe into contact with said membrane thereby binding said probe to said macromolecule;

(d) introducing an enzyme into said chamber device by said fluid delivery means and bringing said enzyme into contact with said probe thereby binding the enzyme to said binding moiety on said probe;

(e) introducing a fluorogenic substrate into said chamber device by said fluid delivery means and bringing said substrate into contact with said enzyme, wherein said enzyme converts said fluorogenic substrate into a fluorescent product and wherein the fluorescent product interacts with the membrane and is spatially localized thereon;

(f) illuminating said fluorescent product in said chamber device with a beam of light from said imaging means capable of exciting fluorescence by said fluorescent product, wherein said fluorescence produces a pattern of binding that reflects the location on said membrane of said macromolecule; and

(g) imaging said pattern of binding by said imaging means, wherein said pattern of binding is stored as digital signals.

79. The method of claim 78 wherein said macromolecule is a nucleic acid.

80. The method of claim 79 wherein step (a) is selected from the group consisting of colony hybridization, plaque hybridization, Southern hybridization, Northern hybridization, multiplex genotyping of simple sequence repeats, sequencing by hybridization, gene mapping by hybridization, dothybridization, slot hybridization, and allele-specific oligonucleotide blot hybridization.

81. The method of claim 78 wherein said macromolecule is a protein.

82. The method of claim 81 wherein step (a) comprises Western blotting.