Automated method for setting up multiple crystallization experiments in submicroliter volumes

• US 7,015,041 B2
• Filed: 12/31/2002
• Issued: 03/21/2006
• Est. Priority Date: 06/18/1999
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Schultz, Peter G., Stevens, Raymond C., Jaklevic, Joseph M., Cornell, Earl W., Yegian, Derek T., Nordmeyer, Robert A., Santarsiero, Bernard D.
• Primary Examiner(s): Gakh, Yelena G.
• Application Number:
  US10/334,336
• Publication Number:
  US 20030119048A1
• Time in Patent Office: 1,176 Days
• Field of Search: 436/4, 436/179, 436/180
• US Class Current: 436/4
• 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 2219/00313 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 3/021 : Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
• 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: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/25625 : Dilution
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method is provided 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 by taking images of the drops.

Claims

1. An automated method for setting up protein crystallization experiments on a multiwell plate comprising:

providing a multiwell plate having a plate surface contoured to define at least 48 different protein crystallization experiment regions, each protein crystallization experiment region comprising a well region housing a mother liquor and a sitting drop region for performing a protein crystallization experiment;

automated delivering of submicroliter portions of the mother liquor from well regions to sitting drop regions of the multiwell plate such that submicroliter portions are delivered to multiple different sitting drop regions of the multiwell plate at the same time; and

automated delivering of submicroliter volumes of a solution containing a protein to be crystallized to the corresponding sitting drop regions of the multiwell plate,

wherein the submicroliter volumes of the solution containing the protein to be crystallized and the submicroliter portions of the mother liquor delivered to the sitting drop regions combine to form protein crystallization solutions of volumes less than 250 nanoliters capable of forming protein crystals suitable for X-ray diffraction;

wherein the multiwell plate being configured such that the remaining mother liquor solution in the well region and the protein crystallization solution in the sitting drop region of each protein crystallization experiment region can be placed in vapor diffusion communication with each other but not in vapor diffusion communication with other protein crystallization regions of the multiwell plate, and

wherein automated delivering of the submicroliter portions of mother liquor and automated delivering of the submicroliter volumes of the solution containing the protein to be crystallized can occur in any order.

2. An method according to claim 1, wherein the mother liquor drop station delivers mother liquor to at least four wells of the multiwell plate at the same time.

3. An method according to claim 1, wherein the mother liquor drop station delivers mother liquor to at least eight wells of the multiwell plate at the same time.

4. A method according to claim 1, wherein the protein crystallization solution forms a drop of a volume less than about 25 nanoliters.

5. A method according to claim 1, wherein the protein crystallization solution forms a drop of a volume less than about 20 nanoliters.

6. A method according to claim 1, wherein the protein crystallization solution forms a drop of a volume less than about 15 nanoliters.

7. A method according to claim 1, wherein the molecule drops formed have a volume of between about 1 nanoliter and 250 nanoliters.

8. A method according to claim 1, wherein submicroliter portions of mother liquor are delivered to the multiwell plate before submicroliter volumes of the solution are delivered to the multiwell plate.

9. A method according to claim 1, wherein submicroliter portions of mother liquor are delivered to the multiwell plate after submicroliter volumes of the solution are delivered to the multiwell plate.

10. An automated method for setting up protein crystallization experiments on a plurality of multiwell plates comprising:

automated transferring a plurality of multiwell plates to a crystallization set-up station, each multiwell plate having a plate surface contoured to define at least 48 different protein crystallization experiment regions, each protein crystallization experiment region comprising a well region housing a mother liquor and a sitting drop region for performing a protein crystallization experiment;

for each multiwell plate transferred to the crystallization set-up station,

automated delivering of submicroliter portions of the mother liquor from well regions to sitting drop regions of the multiwell plate such that submicroliter portions are delivered to multiple different sitting drop regions of the multiwell plate at the same time; and

automated delivering of submicroliter volumes of a solution containing a protein to be crystallized to the corresponding sitting drop regions of the multiwell plate,

wherein the submicroliter volumes of the solution containing the protein to be crystallized and the submicroliter portions of the mother liquor delivered to the sitting drop regions combine to form protein crystallization solutions of volumes less than 250 nanoliters capable of forming protein crystals suitable for X-ray diffraction; and

wherein the microtiter plate being configured such that the remaining mother liquor solution in the well region and the protein crystallization solution in the sitting drop region of each protein crystallization experiment region can be placed in vapor diffusion communication with each other but not in vapor diffusion communication with other protein crystallization regions of the multiwell plate.