Methods for the synthesis of arrays of DNA probes

• US 8,030,477 B2
• Filed: 09/20/2006
• Issued: 10/04/2011
• Est. Priority Date: 02/23/1998
• Status: Expired due to Fees

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Sussman, Michael R., Singh-Gasson, Sangeet, Green, Roland, Cerrina, Francesco, Blattner, Frederick R.
• Primary Examiner(s): Forman; Betty
• Application Number:
  US11/524,082
• Publication Number:
  US 20100227780A1
• Time in Patent Office: 1,840 Days
• Field of Search: None
• US Class Current: 536/25.3
• 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 2219/00439 using micromirror arrays
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential 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 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G02B 26/0841 : the reflecting element being moved or deformed by electrostatic means
• CPC Class Codes: G03F 7/2002 : with visible light or UV light, through an original having an opaque pattern on a transparent support, e.g. film printing, projection printing; by reflection of visible or UV light from an original such as a printed image

Abstract

The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing reflective projection optics. The projection optics project a light image onto the active surface of the substrate to deprotect linker molecules thereon. A first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different light image, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto.

Claims

1. A method of projecting a pattern onto a surface comprising the steps of:

(a) providing a substrate with an active surface to which protected linker molecules have been applied;

(b) projecting a two-dimensional light image onto the active surface of the substrate using projection optics comprising reflective optical elements to illuminate pixel sites on the active surface to photodeprotect the linker molecules thereon, wherein the reflective optical elements provide telecentric projection optics and wherein the step of projecting the two-dimensional image onto the active surface of the substrate comprises;

(i) providing a micromirror device comprising a two-dimensional array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two separate positions, and providing signals to the micromirror device to select a pattern of the micromirrors in the two-dimensional array which are to reflect light onto the active surface; and

(ii) projecting light from a light source onto the micromirror array and reflecting the light from the mirrors of the micromirror array onto the projection optics to project the two-dimensional image onto the active surface,

and further wherein the step of projecting the two-dimensional light image does not use a lithographic mask.

2. The method of claim 1, wherein the reflective optical elements comprise a concave mirror and a convex mirror, the concave mirror reflecting light to the convex mirror which reflects the light back to the concave mirror which reflects the light to the active surface where it is imaged.

3. The method of claim 2, wherein the reflective optical elements further comprise a planar mirror which reflects the light from the concave mirror to the active surface.

4. The method of claim 2, wherein the projection optics further comprise a telecentric aperture placed in front of the convex mirror.

5. The method of claim 2, wherein the radius of curvature of the concave mirror is twice that of the convex mirror.

6. A method of synthesizing arrays of DNA probes comprising carrying out the method of projecting a pattern onto a surface according to claim 2, the method further comprising:

(c) providing a fluid containing an appropriate nucleotide base to the active surface of the substrate and binding the nucleotide base to the photodeprotected linker molecules.

7. The method of claim 1, wherein the substrate is transparent and the light image is projected through a surface of the substrate that is opposite to the active surface.

8. The method of claim 1, further comprising collimating the light from the light source to provide a collimated beam projected onto the micromirror array at an oblique angle to a main optical axis that extends from the micromirror array to the substrate, and wherein in one position of each micromirror the light is reflected along the main optical axis onto the projection optics and in a second position of each micromirror the light from the source is reflected at an angle off the main optical axis and away from the substrate.

9. The method of claim 1, further comprising filtering the light from the light source, whereby only desired wavelengths of light are passed through to the micromirror array.

10. The method of claim 9, wherein the light from the light source comprises ultraviolet or near-ultraviolet light and the desired wavelengths that are passed are in the range of ultraviolet or near-ultraviolet wavelengths.

11. The method of claim 9, wherein the light from the light source is filtered by a filter comprising a dichroic mirror that reflects the desired wavelengths and passes wavelengths to be blocked.

12. The method of claim 1, further comprising a computer connected to the micromirror device to provide command signals to control the deflection of the mirrors in the micromirror array to provide a desired pattern for projection onto the active surface.

13. The method of claim 1, wherein the image that is patterned onto the active surface of the substrate is reduced in size with respect to the size of the array of micromirrors.

14. A method of synthesizing arrays of DNA probes comprising carrying out the method of projecting a pattern onto a surface according to claim 1, the method further comprising:

(c) providing a fluid containing an appropriate nucleotide base to the active surface of the substrate and binding the nucleotide base to the photodeprotected linker molecules.

15. The method of claim 14, further comprising:

(d) projecting a new two-dimensional light image onto the active surface of the substrate to illuminate pixel sites on the active surface to photodeprotect linker molecules or bound nucleotide bases; and

(e) providing a fluid containing an appropriate nucleotide base to the active surface of the substrate and binding the nucleotide base to the photodeprotected linker molecules or bound nucleotide bases.

16. The method of claim 15, wherein steps (d) and (e) are repeated a selected number of times to build up a selected number of levels of nucleotide bases in a DNA probe array on the substrate.

17. The method of claim 14, wherein the nucleotide base is flowed over the active surface in step (c) and bound to the linker molecules utilizing phosphoramidate DNA synthesis.

18. The method of claim 14, wherein the active surface of the substrate is enclosed in a flow cell comprising a sealed reaction chamber, an input port and an output port.

19. The method of claim 18, wherein the flow cell comprises a housing comprising a lower base, an upper cover section and a gasket mounted on the base, wherein the substrate is a transparent glass slide secured between the upper cover section and the lower base to define the sealed reaction chamber between the substrate and the lower base that is sealed by the gasket, and channels extending through the housing from the input port to the reaction chamber and from the reaction chamber to the output port, the active surface of the substrate facing the sealed reaction chamber.