Method and apparatus for fabricating replicate arrays of nucleic acid molecules

• US 6,309,828 B1
• Filed: 11/18/1998
• Issued: 10/30/2001
• Est. Priority Date: 11/18/1998
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Leonard, Leslie A., Schleifer, Arthur, Caren, Michael P., Hotz, Charles Z., Perbost, Michel G. M.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/195,421
• Time in Patent Office: 1,077 Days
• Field of Search: 536/23.1, 536/25.3, 435/6, 435/91.1, 435/287.8, 435/288.3, 435/288.4, 435/288.5, 435/288.6
• US Class Current: 435/6.11
• 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 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00423 using filtration, e.g. through porous frits
• CPC Class Codes: B01J 2219/00452 Means for the recovery of reactants or products
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• 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 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0829 : Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2400/0439 : ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 3/0258 : using stamps
• CPC Class Codes: B01L 3/0262 : using touch-off at substrate or container
• 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 3/0275 : Interchangeable or disposable dispensing tips
• CPC Class Codes: B01L 3/50255 : Multi-well filtration
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

A method and apparatus for fabricating replicate arrays of nucleic acid molecules include the preparation of the molecules and the application the molecules onto a substrate in an ordered array. The apparatus comprises a synthesis unit and a plurality of outlets. The synthesis unit comprises a plurality of synthesis chambers that are spatially arranged relative to each other to provide an array suitable for conducting parallel nucleic acid syntheses. The chambers are suitable for containing discrete compositions of nucleic acid molecules. Each outlet of the plurality of outlets communicates with a single synthesis chamber. The plurality of outlets are configured such that nucleic acid molecules can be removed from the chambers through the outlet and deposited onto the substrate in an ordered array that corresponds to the spatial arrangement of the synthesis chambers.

Claims

1. A device for applying an array of nucleic acid molecules onto a substrate, said device comprising:

(a) a synthesis unit comprising a plurality of synthesis chambers for containing discrete compositions of nucleic acid molecules, wherein the synthesis chambers are spatially arranged relative to each other to provide an array suitable for conducting parallel nucleic acid syntheses; and

(b) a plurality of synthesis outlet means, each of said synthesis outlet means communicating with a single synthesis chamber of said synthesis unit and configured such that the nucleic acid molecules can be removed from said chambers through said synthesis outlet means and deposited onto the substrate without a change in the spatial arrangement in an ordered array that corresponds to the spatial arrangement of the synthesis chambers; and

(c) a purification unit in communication with the synthesis unit, wherein the purification unit comprises a plurality of purification chambers with corresponding purification outlet means in a spatial format, and further wherein the spatial format of the purification chambers is integral with the spatial arrangement of the synthesis unit, such that the synthesized nucleic acid molecules can be transferred to the purification unit without a change in the spatial arrangement.

2. The device of claim 1, wherein each of said synthesis chambers further comprises inlet means.

3. The device of claim 2 wherein the inlet means are comprised of a combination of inkjet components or flow-through methodology.

4. The device of claim 2, wherein the synthesis chambers further comprise a reactive surface on which nucleic acid molecules may be synthesized.

5. The device of claim 4, wherein the reactive surface comprises controlled pore glass (CPG).

6. The device of claim 1, wherein the synthesis outlet means further comprise partitioning means for separating the nucleic acid molecules of different lengths as they pass through the outlet means from the synthesis chambers.

7. The device of claim 1, wherein the outlet means comprise a plurality of microcapillaries, disposable pipette tips or positive displacement techniques.

8. The device of claim 1 further comprising a printing unit in communication with the synthesis unit, wherein the printing unit comprises a plurality of printing chambers with corresponding printing outlet means in a spatial format, and further wherein the spatial format of the printing chambers is integral with the spatial arrangement of the synthesis unit, such that the synthesized nucleic acid molecules can be transferred to the printing unit without a change in the spatial arrangement.

9. The device of claim 8, wherein the printing unit is a rubber stamp, an inkjet device or a capillary deposition unit.

10. The device of claim 8, wherein the printing unit is an inkjet device.

11. The device of claim 8 further comprising means for moving the printing unit toward and away from the substrate.

12. The device of claim 8 wherein the purification unit performs a desalting, chromatographic separation, a separation based on charge or specificity, or a separation of nucleic acid molecules of different lengths.

13. The device of claim 1 wherein the purification chamber comprises a reverse phase chromatography column.

14. The device of claim 1 wherein the purification unit performs a desalting, chromatographic separation, a separation based on charge or specificity, or a separation of nucleic acid molecules of different lengths.

15. A device for applying an array of nucleic acid molecules onto a substrate, said device comprising:

(a) a synthesis unit;

(b) a purification unit; and

(c) a printing unit;

wherein the synthesis unit comprises a plurality of synthesis chambers each having a synthesis outlet means, and wherein the synthesis chambers are spatially arranged relative to each other to provide an array suitable for conducting parallel nucleic acid syntheses and each of said synthesis outlet means communicates with a single synthesis chamber and is configured such that nucleic acid molecules can be removed from said synthesis chambers through said synthesis outlet means and transferred to the purification unit without a change in the spatial arrangement;

and further wherein the purification unit comprises a plurality of purification chambers each having apurification outlet means in a first spatial format, wherein the spatial format of the purification chambers is integral with the spatial arrangement of the synthesis unit, and wherein each of said purification outlet means communicates with a single purification chamber and is configured such that purified nucleic acid molecules can be removed from said purification chambers through said purification outlet means and transferred to the printing unit without a change in the spatial format;

and further wherein the printing unit comprises a plurality of printing chambers each having a printing outlet means in a second spatial format, wherein the spatial format of the printing chambers is integral with the spatial arrangement of the synthesis unit and the spatial format of the purification unit, and wherein each of said printing outlet means communicates with a single printing chamber and is configured such that the purified nucleic acid molecules can be removed from said printing chambers through said printing outlet means and deposited onto a substrate without a change in either spatial formats in an ordered array that corresponds to the spatial arrangement of the synthesis chambers.

16. The device of claim 15 wherein the purification unit performs a desalting, chromatographic separation, a separation based on charge or specificity, or a separation of nucleic acid molecules of different lengths.

17. A method for applying an array of nucleic acid molecules onto a substrate comprising:

(a) providing a device for applying said array of nucleic acid molecules onto a substrate, said device comprising;

(i) a synthesis unit comprising a plurality of synthesis chambers for containing discrete compositions of nucleic acid molecules, wherein the synthesis chambers are spatially arranged relative to each other to provide an array suitable for conducting parallel nucleic acid syntheses;

(ii) a plurality of outlet means, each of said outlet means communicating with a single synthesis chamber of said synthesis unit and the plurality of outlet means configured in a spatial format such that the nucleic acid molecules can be removed from said chambers through said outlet means, wherein the spatial format of the outlet means is integral with the spatial arrangement of the synthesis chamhers; and

(b) treating a reactive surface within each of said synthesis chambers with reagents to yield the nucleic acid molecules in the synthesis chambers;

(c) cleaving said nucleic acid molecules off the reactive surface to yield a plurality of discrete compositions of the nucleic acid molecules; and

(d) depositing the nucleic acid molecules from the chambers onto a substrate without a change in the spatial format in an ordered array that corresponds to the spatial arrangement of the synthesis chambers;

the method further comprising performing post-synthesis purification of the nucleic acid molecules without a change in spatial format prior to deposition onto the substrate.

18. The method of claim 17, wherein the post-synthesis purification comprises a chromatographic separation, a separation based on charge or specificity, or a separation of nucleic acid molecules of different lengths.

19. The method of claim 18, wherein the nucleic acid molecules are deposited onto the substrate by direct contact or positive displacement techniques.

20. The method of claim 19, wherein the nucleic acid molecules are deposited on more than one substrate.

21. The method of claim 20 wherein each of said substrates comprise more than one array patterns.

22. The method of claim 17 wherein the spatial arrangement of the synthesis chambers is integral with that of the array.

23. The method according to claim 17 wherein the depositing of step (d) is performed by a printing unit with outlets, the method additionally comprising the steps of moving the printing unit a distance which is less than the spacing of the outlets and repeating step (d) so that the final array spacing is more dense than the spacing of the outlets.

24. A method for applying an array of nucleic acid molecules onto a substrate comprising:

(a) providing a device for applying said array of nucleic acid molecules onto a substrate, said device comprising;

(i) a synthesis unit comprising a plurality of synthesis chambers for containing discrete compositions of nucleic acid molecules, wherein the synthesis chambers are spatially arranged relative to each other to provide an array suitable for conducting parallel nucleic acid syntheses;

(ii) a plurality of outlet means, each of said outlet means communicating with a single synthesis chamber of said synthesis unit and the plurality of outlet means configured in a spatial format such that the nucleic acid molecules can be removed from said chambers through said outlet means, wherein the spatial format of the outlet means is integral with the spatial arrangement of the synthesis chambers; and

(b) treating a reactive surface within each of said synthesis chambers with reagents to yield the nucleic acid molecules in the synthesis chambers;

(c) cleaving said nucleic acid molecules off the reactive surface to yield a plurality of discrete compositions of the nucleic acid molecules; and

(d) depositing the nucleic acid molecules from the chambers onto a substrate without a change in the spatial format in an ordered array that corresponds to the spatial arrangement of the synthesis chambers;

wherein the treating of step (b) comprises;

(i) deprotecting a protected monomer coupled to the surface, (ii) adding an additional protected monomer to the synthesis chamber to couple to the deprotected monomer, and (iii) repeating steps (i) and (ii) to yield the nucleic acid molecules;

the method further comprising performing post-synthesis purification of the nucleic acid molecules without a change in spatial format prior to deposition onto the substrate.

25. The method of claim 24, wherein the post-synthesis purification comprises a chromatographic separation, a separation based on charge or specificity, or a separation of nucleic acid molecules of different lengths.

26. The method of claim 24 wherein the depositing is performed by an inkjet device.

27. The method of claim 24 wherein the spatial arrangement of the synthesis chambers is integral with that of the array.

28. The method of claim 24 wherein the depositing of step (d) is performed by a printing unit with outlets, the method additionally comprising the steps of moving the printing unit a distance which is less than the spacing of the outlets and repeating step (d) so that the final array spacing is more dense than the spacing of the outlets.

29. The method of claim 24 wherein the treating step additionally comprising washing to remove excess reagents following addition of the additional protected monomer to the synthesis chamber to couple to the deprotected monomer.