Whole genome expression analysis system

• US 20050233363A1
• Filed: 03/31/2005
• Published: 10/20/2005
• Est. Priority Date: 09/19/2003
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Kao, H. Pin, Harding, Ian A., Fawcett, Adrian, Oldham, Mark, Jones, Robert C., Woudenberg, Tim, Bodeau, John P., Bodner, Kevin, Sandell, Donald, Lim, Gary
• Application Number:
  US11/096,282
• Publication Number:
  US 20050233363A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 2300/041 Connecting closures to device or container
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/50853 with covers or lids
• 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: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening

Abstract

A method for simultaneously determining a genetic expression profile for an individual member of a species relative to an entire standard genome for the species. The method can comprise distributing a liquid sample into an array of reaction chambers of a substrate. The array can comprise a primer set and a probe for each polynucleotide target along the entire standard genome. The liquid sample can comprise substantially all genetic material of the member. Each of the reaction chambers can comprise the primer set and the probe for at least one of the polynucleotide targets and a polymerase. The method can further comprise amplifying the liquid sample in the array, detecting a signal emitted by at least one of the probes, and identifying the genetic expression profile in response to the signal.

Claims

1. A method for simultaneously determining a genetic expression profile for an individual member of a species relative to an entire standard genome for the species, said method comprising:

distributing a liquid sample into an array of reaction chambers of a substrate, said array having a primer set and a probe for each of a plurality of polynucleotide targets for the entire standard genome, said liquid sample having substantially all genetic material of said member, each of said reaction chambers having said primer set and said probe for at least one of said plurality of polynucleotide targets and a polymerase;

amplifying said liquid sample in said array;

detecting a signal emitted by at least one of said probes; and

identifying said genetic expression profile in response to said signal.

2. The method according to claim 1 wherein said amplifying said liquid sample in said array includes amplifying said liquid sample using PCR.

3. The method according to claim 2 wherein said amplifying said liquid sample using PCR includes amplifying said liquid sample using real time PCR.

4. The method according to claim 2 wherein said amplifying said liquid sample using PCR includes amplifying said liquid sample using multiplex PCR.

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

forcing said liquid sample into said reaction chambers after said distributing said liquid sample into said array of reaction chambers.

6. The method according to claim 5 wherein said forcing said liquid sample into said reaction chambers includes forcing said liquid sample into said reaction chambers using centrifugal force.

7. The method according to claim 5 wherein said forcing said liquid sample into said reaction chambers includes forcing said liquid sample into said reaction chambers using pneumatic pressure.

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

preamplifying said liquid sample before said distributing said liquid sample into said array of reaction chambers.

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

sealing each of said reaction chambers with a sealing cover.

10. The method according to claim 1 wherein said distributing said liquid sample comprises:

flooding a surface of said substrate with said liquid sample.

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

distributing amplification reactants to said reaction chambers.

12. The method according to claim 1 wherein said distributing said liquid sample comprises spraying a surface of said substrate with said liquid sample.

13. The method according to claim 1 wherein said distributing said liquid sample into said array of reaction chambers includes distributing said liquid sample into said array of reaction chambers having a volume of less than about 1 microliter.

14. A microplate assembly for simultaneously determining a genetic expression profile for an individual member of a species relative to an entire standard genome for the species, said microplate assembly comprising:

a substrate;

at least about 1000 reaction chambers formed in an array on said substrate, said array having a primer set and a probe for each of a plurality of polynucleotide targets within the entire standard genome, each of said reaction chambers having said primer set and said probe for at least one of said plurality of polynucleotide targets and a polymerase; and

a cover.

15. The microplate assembly according to claim 14 wherein each of said reaction chambers has a volume less than about 50 microliters.

16. The microplate assembly according to claim 14 wherein each of said reaction chambers has a volume less than about 20 microliters.

17. The microplate assembly according to claim 14 wherein said entire standard genome is selected from the group consisting essentially of human, mouse, rat, rabbit, primate, bacteria, plant, insect, dog, fungus, yeast, and virus species.

18. The microplate assembly according to claim 14 wherein said array comprises at least about 6,000 reaction chambers.

19. The microplate assembly according to claim 14, further comprising:

a clamping system coupling said cover to said substrate.

20. The microplate assembly according to claim 14 wherein a distance between a first of said reaction chambers and an adjacent one of said reaction chambers is about 50 to about 500 microns.

21. The microplate assembly according to claim 14 wherein said substrate has a width of about 10 to about 200 mm and a length of about 10 to about 200 mm.

22. The microplate assembly according to claim 14 wherein a depth of each of said reaction chambers is about 800 to about 3000 microns.

23. The microplate assembly according to claim 14, further comprising:

a filling system operably coupled with said substrate.

24. The microplate assembly according to claim 23 wherein said filling system comprises:

a trench formed in a surface of said substrate.

25. The microplate assembly according to claim 23 wherein said filling system comprises:

a gasket mounted between said cover and said substrate, said gasket creating a space between said cover and said substrate operable to carry a liquid.

26. The microplate assembly according to claim 25 wherein said gasket substantially seals said space between said cover and said substrate, said gasket having at least one input port.

27. The microplate assembly according to claim 14, further comprising:

a sealing adhesive disposed between said cover and said substrate for sealing said cover to said substrate.

28. The microplate assembly according to claim 14 wherein said entire standard genome comprises greater than about 20,000 polynucleotide targets.

29. A system for simultaneously determining a genetic expression profile in a liquid sample obtained from an individual member of a species relative to an entire standard genome for the species, said system comprising:

a substrate;

at least about 1000 reaction chambers formed in an array on said substrate, said array having a primer set and a probe for each of a plurality of polynucleotide targets within the entire standard genome, each of said reaction chambers having said primer set and said probe for at least one of said plurality of polynucleotide targets and a polymerase;

a cover;

amplification reagents for performing an amplification reaction;

a filling system in fluid communication with each of said reaction chambers for filling the liquid sample and said amplification reagents into each of said reaction chambers;

a thermal cycling system in thermal communication with each of said reaction chambers for controlling a temperature during said amplification reaction;

an excitation system disposed proximate said reaction chambers to excite at least one of said probes to emit a signal; and

a detection system detecting said signal.

30. The system according to claim 29 wherein each of said reaction chambers has a volume less than about 50 microliters.

31. The system according to claim 29 wherein each of said reaction chambers has a volume less than about 20 microliters.

32. The system according to claim 29 wherein said entire standard genome is selected from the group consisting essentially of human, mouse, dog, rat, rabbit, primate, bacteria, plant, insect, fungus, yeast and virus species.

33. The system according to claim 29 wherein said entire standard genome comprises:

at least about 20,000 polynucleotide targets.

34. The system according to claim 29 wherein said substrate comprises:

at least about 20,000 reaction chambers.

35. The system according to claim 29 wherein said amplification reaction is real time polymerase chain reaction.

36. The system according to claim 29 wherein a distance between a first of said reaction chambers and a second of said reaction chambers is about 50 to about 500 microns.

37. The system according to claim 29, further comprising:

a clamping system operably coupling at least one of said substrate and said cover onto said thermal cycling system.

38. The system according to claim 29 wherein said substrate has a width of about 10 to about 200 mm and a length of about 10 to about 200 mm.

39. The system according to claim 29 wherein a depth of each of said reaction chambers is about 800 to about 3000 microns.

40. The system according to claim 29 wherein said excitation system includes an array of light emitting diodes.

41. The system according to claim 29 wherein said detection system includes a high resolution CCD camera.

42. The system according to claim 29 wherein said substrate is made of a thermally conductive material.

43. The system according to claim 29, further comprising:

a microprocessor for analyzing said signal to determine a genetic expression profile.

44. The system according to claim 29, further comprising:

a database holding information relating to said entire standard genome in a media.

45. The system according to claim 44, further comprising:

a web portal connecting at least one microprocessor with said database.

46. A method for simultaneously determining a genetic expression profile for an individual member of a species relative to an entire standard genome for the species, said method comprising:

distributing a liquid sample into an array of material retention regions of a substrate, said array having a primer set and a probe for each of a plurality of polynucleotide targets for the entire standard genome, said liquid sample having substantially all genetic material of said member, each of said material retention regions having said primer set and said probe for at least one of said plurality of polynucleotide targets and a polymerase;

sealing said array of material retention regions with a cover;

inverting said array of material retention regions such that said liquid sample contacts said cover;

amplifying said liquid sample in said array;

detecting a signal emitted by at least one of said probes; and

identifying said genetic expression profile in response to said signal.

47. The method according to claim 46 wherein said amplifying said liquid sample in said array includes amplifying said liquid sample using PCR.

48. The method according to claim 47 wherein said amplifying said liquid sample using PCR includes amplifying said liquid sample using real time PCR.

49. The method according to claim 47 wherein said amplifying said liquid sample using PCR includes amplifying said liquid sample using multiplex PCR.

50. The method according to claim 46, further comprising:

preamplifying said liquid sample before said distributing said liquid sample into said array of material retention regions.

51. The method according to claim 46, further comprising:

distributing amplification reactants to said material retention regions.

52. The method according to claim 46, further comprising:

forcing said liquid sample into said material retention regions after said distributing said liquid sample into said array of material retention regions.

53. The method according to claim 52 wherein said forcing said liquid sample into said material retention regions includes forcing said liquid sample into said material retention regions using centrifugal force.

54. The method according to claim 52 wherein said forcing said liquid sample into said material retention regions includes forcing said liquid sample into said material retention regions using pneumatic pressure.

55. The method according to claim 46 wherein said distributing said liquid sample into said array of material retention regions includes distributing said liquid sample into said array of material retention regions having a volume of less than about 1 microliter.