Capillary array-based sample screening

• US 6,794,127 B1
• Filed: 10/12/2000
• Issued: 09/21/2004
• Est. Priority Date: 06/16/1997
• Status: Expired due to Term

• Current Assignee: BASF Enzymes LLC (BASF SE)
• Original Assignee: Diversa Corporation (BASF SE)
• Inventors: Keller, Martin, Short, Jay M., Lafferty, William Michael
• Primary Examiner(s): Nashed, Nashaat T.
• Application Number:
  US09/687,219
• Time in Patent Office: 1,440 Days
• Field of Search: 435/6, 435/4, 435/18
• US Class Current: 435/4
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00367 capillary
• CPC Class Codes: B01J 2219/00416 Vacuum
• CPC Class Codes: B01J 2219/00479 Means for mixing reactants or products in the reaction vessels
• CPC Class Codes: B01J 2219/00481 by the use of moving stirrers within the reaction vessels
• CPC Class Codes: B01J 2219/00522 in a multiple parallel arrangement
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00702 : Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/0074 : Biological products
• CPC Class Codes: B01J 2219/00743 : Cells
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50857 : using arrays or bundles of open capillaries for holding samples
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/1037 : Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1055 : Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12Q 1/6811 : Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C40B 40/02 : Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

Provided are methods of screening and identification of bio activities and bioactive molecules of interest using a capillary array system. More specifically, disclosed are methods of using optical detection and capillary array-based techniques for screening libraries and recovering bioactive molecules having a desired activity or a nucleic acid sequence encoding such bioactive molecules.

Claims

1. A method for identifying an enzymatic activity of interest, comprising:

(a) introducing a fluorogenic or chromogenic substrate and a recombinant clone into a capillary tube of a capillary array, wherein each capillary tube of the capillary array comprises at least one wall defining a lumen for retaining the substrate and the recombinant clone, and wherein the at least one wall is comprised of a material having a low refractive index;

(b) culturing the clone in the capillary tube containing the substrate and the recombinant clone under conditions which allow interaction of the substrate and an enzyme produced by the recombinant clone to produce a detectable optical signal; and

(c) detecting the detectable optical signal in the capillary tube to identify one or more capillaries containing the detectable optical signal, thereby identifying the enzymatic activity of interest.

2. The method of claim 1, wherein the recombinant clone is from a library of clones.

3. The method of claim 2, wherein the library is an expression library.

4. The method of claim 3, wherein the expression library is generated from genomic DNA of one or more microorganisms.

5. The method of claim 4, wherein the microorganisms are marine microorganisms.

6. The method of claim 4, wherein the microorganisms are airborne microorganisms.

7. The method of claim 4, wherein the microorganisms comprise prokaryotic cells.

8. The method of claim 4, wherein the microorganisms are derived from an environmental sample.

9. The method of claim 4, wherein the microorganisms are terrestrial microorganisms.

10. The method of claim 4, wherein the microorganisms comprise extremophiles.

11. The method of claim 10, wherein the extremophiles are thermophiles.

12. The method of claim 10, wherein the extremophiles are selected from the group consisting of hyperthernophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles and acidophiles.

13. The method of claim 4, wherein the host cells are selected from the group consisting of bacterial cells, fungal cells, plant cells, insect cells and animal cells.

14. The method of claim 4, wherein the host cells are prokaryotic cells.

15. The method of claim 14, wherein the prokaryotic cells are bacterial cells.

16. The method of claim 15, wherein the bacterial cells are E. coli, Pseudomonas, or Bacillus.

17. The method of claim 1, wherein the enzyme is a hydrolase.

18. The method of claim 1, wherein the detectable optical signal is a change in color.

19. The method of claim 1, wherein the detectable optical signal is a change in fluorescence.

20. The method of claim 19, wherein the substrate is selected from the group consisting of umbelliferone, resorufin, fluorescein rhodamine, and derivatives thereof.

21. The method of claim 1, wherein the detection is provided by a detector comprising a photodetector.

22. The method of claim 1, wherein the capillary array comprises at least about 100 capillary tubes.

23. The method of claim 1, wherein the capillary array comprises at least about 1000 capillary tubes.

24. The method of claim 1, wherein the capillary array comprises at least about 5000 capillary tubes.

25. The method of claim 1, wherein the capillary array comprises at least about 100,000 to about 4,000,000 capillary tubes.

26. The method of claim 1, wherein the capillary array has a capillary tube density of about 500 capillary tubes/cm² to 51,000 capillary tubes/cm².

27. The method of claim 1, wherein the height:

width aspect ratio of the capillary army is at least about 1.

28. The method of claim 1, wherein the substrate and the recombinant clone are introduced simultaneously as a cell/substrate mixture into capillary tubes in the capillary army.

29. The method of claim 1, further comprising biopanning prior to step (a).

30. The method of claim 4, further comprising normalizing the genomic DNA samples prior to generating the library.

31. The method of claim 1, further comprising the step of isolating one or more enzymes from the recovered contents of the one or more capillary tubes.

32. The method of claim 1, further comprising recovering and sequencing a polynucleotide from the recombinant clone encoding the desired enzymatic activity.

33. The method of claim 1, wherein the substrate comprises C12FDG.

34. The method of claim 1, further comprising after (c):

(d) recovering the contents of the capillary tube containing the detectable signal, thereby recovering the enzymatic activity of interest.

35. The method of claim 1, wherein the enzyme is a lipase.

36. The method of claim 1, wherein the enzyme is a hydratase

37. The method of claim 1, wherein the enzyme is an esterase.

38. The method of claim 1, wherein the enzyme is a protease.

39. The method of claim 1, wherein the enzyme is a peptidase.

40. The method of claim 1, wherein the enzyme is a reductase.

41. The method of claim 1, wherein the enzyme is an oxidoreductase.

42. The method of claim 1, wherein the enzyme is a lyase.

43. The method of claim 1, wherein the enzyme is a ligase.

44. The method of claim 1, wherein the enzyme is an isomerase.

45. The method of claim 1, wherein the enzyme is a polymerase.

46. The method of claim 1, wherein the enzyme is a synthetase.

47. The method of claim 1, wherein the enzyme is a glycosidase.

48. The method of claim 1, wherein the enzyme is a transferase.

49. The method of claim 1, wherein the enzyme is a phosphatase.

50. The method of claim 1, wherein the enzyme is a kinase.

51. The method of claim 1, wherein the enzyme is a mono- or di-oxygenase.

52. The method of claim 1, wherein the enzyme is a nitrilase.

53. The method of claim 1, wherein the enzyme is a peroxidase.

54. The method of claim 1, wherein the enzyme is a transaminase.

55. The method of claim 1, wherein the enzyme is an amidase.

56. The method of claim 1, wherein the enzyme is an acylase.