Methods and apparatus for performing array microcrystallizations

• US 6,296,673 B1
• Filed: 06/18/1999
• Issued: 10/02/2001
• Est. Priority Date: 06/18/1999
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California), University The Regents of The California
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Jin, Jian, Cornell, Earl, Kolbe, William F., Shultz, Peter G., Santarsiero, Bernard D., Jones, Arthur L., Uber, Donald C., Stevens, Raymond C., Jaklevic, Joseph M., Yegian, Derek T., Nordmeyer, Robert A.
• Primary Examiner(s): Griffin, Steven P.
• Assistant Examiner(s): MEDINA SANABRIA, MARIBEL
• Application Number:
  US09/336,134
• Time in Patent Office: 837 Days
• Field of Search: 232/95.R, 436/86, 436/174
• US Class Current: 23/295.00R
• 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 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00328 Movement by linear translation
• CPC Class Codes: B01J 2219/00376 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00412 In multiple arrangements
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0065 by the use of liquid beads
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00756 Compositions, e.g. coatings, crystals, formulations
• CPC Class Codes: B01L 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 2300/04 Closures and closing means
• CPC Class Codes: B01L 2300/0609 Holders integrated in container to position an object
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/10 Means to control humidity and/or other gases
• CPC Class Codes: B01L 2400/0478 : pistons
• CPC Class Codes: B01L 2400/0633 : with moving parts
• CPC Class Codes: B01L 3/0268 : using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50851 : specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/5088 : confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C07K 2299/00 : Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: C30B 29/58 : Macromolecular compounds
• CPC Class Codes: C30B 7/00 : Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions from molten solvents C30B9/00; by normal or gradient freezing C30B11/00; under a protective fluid C30B27/00
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: C40B 70/00 : Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 2001/4027 : evaporation leaving a concentrated sample
• CPC Class Codes: Y10T 436/25 : including sample preparation

Abstract

A method is provided for performing array microcrystallizations to determine suitable crystallization conditions for a molecule. The method comprises forming an array of microcrystallizations, each microcrystallization comprising a drop containing a molecule to be crystallized, the drop having a volume of less than 1 μL; storing the array of microcrystallizations under conditions suitable for molecule crystals to form in the drops in the array; and detecting molecule crystal formation in the drops.

Claims

1. A method for performing array microcrystallizations to determine suitable crystallization conditions for a molecule, the method comprising:

forming an array of microcrystallizations, each microcrystallization comprising a drop comprising a mother liquor solution whose composition varies within the array and a molecule to be crystallized, the drop having a volume of less than 1 microliter;

storing the array of microcrystallizations under conditions suitable for molecule crystals to form in the drops in the array; and

detecting molecule crystal formation in the drops.

2. The method according to claim 1 wherein the molecule is a macromolecule.

3. The method according to claim 1 wherein the molecule is a protein.

4. The method according to claim 1 wherein the macromolecule has a molecular weight of at least 500 daltons.

5. The method according to claim 1 wherein the drop has a volume of less than about 750 nL.

6. The method according to claim 1 wherein the drop has a volume of less than about 500 nL.

7. The method according to claim 1 wherein the drop has a volume of less than about 250 nL.

8. The method according to claim 1 wherein the drop has a volume of between about 1 nL-750 nL.

9. The method according to claim 1 wherein the drop has a volume of between about 1 nL-500 nL.

10. The method according to claim 1 wherein the drop has a volume of between about 1 nL-250 nL.

11. The method according to claim 1 wherein each microcrystallization further includes a volume of mother liquor solution separate from the drop, the mother liquor solution contained in the volume having the same composition as the mother liquor solution contained in the drop, the volume comprising less than about 500 microliters of the mother liquor solution.

12. The method according to claim 1 wherein each microcrystallization further includes a volume of mother liquor solution separate from the drop, the mother liquor solution contained in the volume having the same composition as the mother liquor solution contained in the drop, the volume comprising less than about 250 microliters of the mother liquor solution.

13. The method according to claim 1 wherein the mother liquor solutions have at least 4 components which are varied within the array.

14. The method according to claim 1 wherein the mother liquor solutions have at least 5 components which are varied within the array.

15. The method according to claim 1 wherein the array includes greater than 96 microcrystallizations.

16. The method according to claim 1 wherein the array includes greater than 192 microcrystallizations.

17. The method according to claim 1 wherein forming the array of microcrystallizations includes using greater than 48 stock solutions to form the mother liquor solutions used in the array.

18. The method according to claim 1 wherein forming the array of microcrystallizations includes using greater than 96 stock solutions to form the mother liquor solutions used in the array.

19. The method according to claim 1 wherein forming the array of microcrystallizations includes using greater than 192 stock solutions to form the mother liquor solutions used in the array.

20. The method according to claim 1 wherein forming the array of microcrystallizations includes forming the drops within a volume range of less than about 25 nL.

21. The method according to claim 1 wherein forming the array of microcrystallizations includes forming the drops within a volume range of less than about 20 nL.

22. The method according to claim 1 wherein forming the array of microcrystallizations includes forming the drops within a volume range of less than about 15 nL.

23. A method for performing array microcrystallizations to determine suitable crystallization conditions for a molecule, the method comprising:

forming an array of microcrystallizations, each microcrystallization comprising a hanging drop comprising a mother liquor solution whose composition varies within the array and a molecule to be crystallized, the drop having a volume of less than 1 microliter;

storing the array of microcrystallizations under conditions suitable for molecule crystals to form in the drops in the array; and

detecting molecule crystal formation in the drops.

24. A method for performing arrat microcrystallizations to determine suitable crystallization conditions for a molecule, the method comprising:

forming an array of microcrystallizations, each microcrystallization comprising a sitting drop comprising a mother liquor solution whose composition varies within the array and a molecule to be crystallized, the drop having a volume of less than 1 microliter;

storing the array of microcrystallizations under conditions suitable for molecule crystals to form in the drops in the array; and

detecting molecule crystal formation in the drops.

25. A method for performing array microcrystallizations to determine suitable crystallization conditions for a molecule, the method comprising:

forming a first array of microcrystallizations, each microcrystallization comprising a drop comprising a mother liquor solution whose composition varies within the array and a molecule to be crystallized, the drop having a volume of less than 1 microliter;

storing the first array of microcrystallizations under conditions suitable for molecule crystals to form in the drops in the array;

detecting molecule crystal formation in the drops;

forming a second array of microcrystallizations, each microcrystallization comprising a drop comprising a molecule to be crystallized and a mother liquor solution whose composition varies within the array and is based on one or more mother liquor solutions used in the first array in which molecule crystals were detected;

storing the second array of microcrystallizations under conditions suitable for molecule crystals to form drops in the array; and

detecting molecule crystal formation in the drops.

26. The method according to claim 25 wherein drop is a hanging drop.

27. The method according to claim 25 wherein drop is a sitting drop.

28. The method according to claim 25 wherein drops formed in the second array have a volume of less than 1 microliter.

29. A method for forming submicroliter drops for use in an array microcrystallization to determine suitable crystallizationi conditions for a molecule, the method comprising:

delivering to a plurality of drop regions within a volume range of less than about 25 nl submicroliter volues of mother liquor solutions whose compositions vary within the array; and

delivering to the plurality of drop regions submicroliter volumes of a solution containing a molecule to be crystallized within a volume range of less than about 25 nL;

wherein a total volume of the submicroliter volumes delivered to each drop region is less than 1 microliter.

30. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region is less than about 750 nL.

31. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region is less than about 500 nL.

32. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region is less than about 250 nL.

33. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region is between about 1 nL-750 nL.

34. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region is between about 1 nL-500 nL.

35. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region between about 1 nL-250 nL.

36. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region is within a volume range of less than about 20 nL.

37. The method according to claim 29 wherein the submicroliter volumes delivered to each drop region is within a volume range of less than about 15 nL.

38. A method for forming submicroliter hanging drops on cover slips for use in an array microcrystallization to determine suitable crystallization conditions for a molecule, the method comprising:

taking a plurality of coverslips;

removing mother liquor from a plurality of wells of a multiwell plate;

delivering submicroliter volumes of the mother liquor to the plurality of coverslips within a volume range of less than about 25 nL; and

delivering submicroliter volumes of a solution containing molecule to be crystallized to the plurality of coverslips within a volume range of less than about 25 nL;

wherein a total volume of the submicroliter volumes delivered to each coverslip is less than 1 microliter.

39. A method for forming a submicroliter sitting drops for use in an array microcrystallization to determine suitable crystallization conditions for a molecule, the method comprising:

taking a multiwell plate comprising a plurality of regions, each region including a sitting drop region and a well comprising liquor solution whose composition varies within the regions of the multiwell plate;

delivering submicroliter volumes of the mother liquor solutions from the wells to the sitting drop regions within a volume range of less than about 25 nL; and

delivering submicroliter volumes of a solution containing a molecule to be crystallized to the sitting drop regions within a volume range of less than about 25 nL;

wherein a total volume of the submicroliter volumes delivered to each sitting drop reguib is less than 1 microliter.

40. A method for performing array microcrystallizations to determine suitable crystallization conditions for a molecule, the method comprising:

forming an array of microcrystallizations, each microcrystallization comprising a microcrystallization volume comprising a mother liquor solution whose composition varies within the array and a molecule to be crystallized, the microcrystallization volume having a volume of less than 1 microliter;

storing the array of microcrystallizations under conditions suitable for molecule crystals to form in the microcrystallization volumes; and

detecting molecule crystal formation in the microcrystallization volumes.

41. The method according to claim 40 wherein the molecule is a macromolecule.

42. The method according to claim 40 wherein the molecule is a protein.

43. The method according to claim 40 wherein the macromolecule has a molecular weight of at least 500 daltons.

44. The method according to claim 40 wherein the microcrystallization volume has a volume of less than about 750 nL.

45. The method according to claim 40 wherein the microcrystalization volume has a volume of less than about 500 nL.

46. The method according to claim 40 wherein the microcrystalization volume has a volume of less than about 250 nL.

47. The method according to claim 40 wherein the microcrystalization volume has a volume of between about 1 nL-750 nL.

48. The method according to claim 40 wherein the microcrystalization volume has a volume of between about 1 nL-500 nL.

49. The method according to claim 40 wherein the microcrystalization volume has a volume of between about 1 nL-250 nL.

50. The method according to claim 40 wherein the mother liquor solutions have at least 4 components which are varied within the array.

51. The method according to claim 40 wherein the mother liquor solutions have at least 5 components which are varied within the array.

52. The method according to claim 40 wherein the array includes greater than 96 microcrystallizations.

53. The method according to claim 40 wherein the array includes greater than 192 microcrystallizations.

54. The method according to claim 40 wherein forming the array of microcrystallizations includes using greater than 48 stock solutions to form the mother liquor solutions used in the array.

55. The method according to claim 40 wherein forming the array of microcrystallizations includes using greater than 96 stock solutions to form the mother liquor solutions used in the array.

56. The method according to claim 40 wherein forming the array of microcrystallizations includes using greater than 192 stock solutions to form the mother liquor solutions used in the array.

57. The method according to claim 40 wherein forming the array of microcrystallizations includes forming the microcrystallization volumes within a volume range of less than about 25 nL.

58. The method according to claim 40 wherein forming the array of microcrystallizations includes forming the microcrystallization volumes within a volume range of less than about 20 nL.

59. The method according to claim 40 wherein forming the array of microcrystallizations includes forming the microcrystallization volumes within a volume range of less than about 15 nL.