Computer-assisted methods and apparatus for identification and characterization of biomolecules in a biological sample

• US 6,064,754 A
• Filed: 12/01/1997
• Issued: 05/16/2000
• Est. Priority Date: 11/29/1996
• Status: Expired due to Fees

• Current Assignee: Oxford Glycosciences (UK) Limited (Financière de Tubize SA)
• Original Assignee: Oxford Glycosciences (UK) Limited (Financière de Tubize SA)
• Inventors: Platt, Albert Edward, Parekh, Rajesh Bhikhu, Bruce, James Alexander, Prime, Sally Barbara, Amess, Robert, Stoney, Richard Michael
• Primary Examiner(s): Johns, Andrew W.
• Application Number:
  US08/980,574
• Time in Patent Office: 897 Days
• Field of Search: 382/128, 382/129, 382/153, 364/528.01, 364/528.08, 702/19, 204/461, 204/607, 204/612, 436/516, 435/7.1, 435/288.7, 700/266, 700/273
• US Class Current: 382/129
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: G01N 2001/288 Filter punches
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01N 27/44739 Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
• CPC Class Codes: G01N 27/44782 of a plurality of samples

Abstract

The present invention provides computer-assisted methods and apparatus for identifying, selecting and characterizing biomolecules in a biological sample. A two-dimensional array is generated by separating biomolecules present in a complex mixture. A computer-readable profile is constructed representing the identity and relative abundance of a plurality of biomolecules detected by imaging the two dimensional array. Computer-mediated comparison of profiles from multiple samples permits automated identification of subsets of biomolecules that satisfy pre-ordained criteria. Identified biomolecules can be automatically isolated from the two dimensional array by a robotic device in accordance with computer-generated instructions. A supported gel suitable for electrophoresis is provided that is bonded to a solid support such that the gel has two-dimensional spatial stability and the solid support is substantially non-interfering with respect to detection of a label, such as a fluorescent label, associated with one or more biomolecules in the gel.

Claims

1. A computer-assisted method for selecting and directing the isolation of one or more biomolecules present in a two-dimensional array, comprising:

imaging said two-dimensional array or a replica thereof to generate a computer-readable output comprising, for each of a plurality of biomolecules detected in said two-dimensional array, a pair of x,y coordinates and a signal value;

processing said output in at least one computer to select one or more of said detected biomolecules in accordance with previously ordained or operator-specified criteria; and

generating machine-readable instructions that direct a robotic device to isolate at least one of said selected biomolecules from said two-dimensional array.

2. The method according to claim 1, further comprising:

isolating at least one of said selected biomolecules from said two-dimensional array by means of said robotic device in accordance with said machine-readable instructions.

3. The method according to claim 2, further comprising, prior to said imaging:

a first separation step, wherein a plurality of biomolecules present in a biological sample are separated according to a first physical or chemical property to form a one-dimensional array of biomolecules;

a second separation step, wherein said one-dimensional array of biomolecules is separated according to a second physical or chemical property to form said two-dimensional array.

4. The method according to claim 3, wherein said first separation step results in a discontinuous one-dimensional array.

5. The method according to claim 1, wherein said machine-readable instructions direct x and y movements of said robotic device.

6. The method according to claim 5, further comprising:

isolating at least one of said selected biomolecules from said two-dimensional array by means of said robotic device in accordance with said machine-readable instructions.

7. The method according to claim 6, further comprising, prior to said imaging:

a first separation step, wherein a plurality of biomolecules present in a biological sample are separated according to a first physical or chemical property to form a one-dimensional array of biomolecules;

a second separation step, wherein said one-dimensional array of biomolecules is separated according to a second physical or chemical property to form said two-dimensional array.

8. The method according to claim 5, wherein said processing is in accordance with previously ordained criteria.

9. The method according to claim 5, wherein said processing is in accordance with operator-specified criteria.

10. The method according to claim 5 or 6, in which said biomolecules are oligosaccharides.

11. The method according to claim 5 or 6, in which said biomolectles are proteins.

12. The method according to claim 11, in which said proteins are glycoproteins.

13. The method according to claim 11, in which said two-dimensional array is contained in a polyacrylamide gel.

14. The method according to claim 13, in which said proteins have been separated by isoelectric focusing, followed by electrophoresis in the presence of sodium dodecyl sulfate.

15. The method according to claim 13, in which said polyacrylamide gel is bonded to a generally planar solid support such that the gel has two-dimensional spatial stability, and the support is substantially non-interfering with respect to detection of a detectable label carried by the proteins.

16. The method according to claim 15, in which said polyacrylamide gel is covalently bonded to said solid support.

17. The method according to claim 16, in which said detectable label is a fluorescent label.

18. The method according to claim 17, in which said solid support is glass.

19. Apparatus for computer-assisted isolation of one or more selected biomolecules present in a two-dimensional array, comprising:

a detector capable of imaging said two-dimensional array to generate a computer-readable output comprising, for each of a plurality of biomolecules in said two-dimensional array, a pair of x,y coordinates and a signal value;

one or more computers programmed to select one or more biomolecules represented in said output in accordance with previously ordained or operator-specified criteria and to generate machine-readable instructions that direct a robotic device to isolate at least one of said selected biomolecules; and

a robotic device capable of isolating at least one of said selected biomolecules in accordance with said instructions,

wherein said detector is operably connected to at least one of said one or more computers, and at least one of said one or more computers is operably connected to said robotic device.

20. The apparatus according to claim 19, wherein said machine-readable instructions direct said robotic device to perform x and y movements and said robotic device is capable of performing x and y movements in accordance with said instructions.

21. The apparatus according to claim 20, in which said separated biomolecules are proteins.

22. The apparatus according to claim 21, in which said two-dimensional array is contained in a polyacrylamide gel.

23. The apparatus according to claim 22, in which said polyacrylamide gel is bonded to a generally planar solid support such that the gel has two-dimensional spatial stability, and the support is substantially non-interfering with respect to detection of a detectable label carried by the proteins.

24. The apparatus according to claim 23, in which said polyacrylamide gel is covalently bonded to said solid support.

25. The apparatus according to claim 23, in which said detectable label is a fluorescent label.

26. The apparatus according to claim 25, in which said solid support is glass.