Amplification and other enzymatic reactions performed on nucleic acid arrays

• US 6,248,521 B1
• Filed: 07/21/1998
• Issued: 06/19/2001
• Est. Priority Date: 07/22/1997
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Qiagen Genomics, Inc.
• Inventors: Van Ness, Jeffrey, Tabone, John C., Moynihan, Kristen
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/120,501
• Time in Patent Office: 1,064 Days
• Field of Search: 435/6, 435/91.2, 435/71.1, 427/333
• US Class Current: 435/6.12
• 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 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• 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 2219/00612 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 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 : Allele-specific amplification
• CPC Class Codes: C12Q 2527/101 : Temperature
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2533/101 : Primer extension
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• 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: G01N 2035/00455 : Controlling humidity in analyser

Abstract

The present invention provide methods and an apparatus for performing amplification and other enzymatic reactions on nucleic acid molecules that have been printed onto a solid substrate, such as a silicon wafer or glass slide.

Claims

1. A method of amplifying nucleic acid molecules from a template in a chamber, comprising:

(a) mixing single-stranded nucleic acid templates on a solid substrate with a solution comprising an oligonucleotide primer that hybridizes to the templates and a DNA polymerase, wherein the mixing occurs in discrete areas on the substrate, and wherein the solution remains in the discrete areas;

(b) synthesizing a complementary strand to the template to form a duplex;

(c) denaturing the duplex; and

(d) synthesizing complementary strands to the template, therefrom amplifying nucleic acid molecules;

wherein the nucleic acid solution is in contact with the atmosphere in the chamber, and dew point is maintained during said mixing, synthesizing, and denaturing, thereby preventing evaporation of the solution.

3. The method of either of claims 1 or 2, wherein steps (c) and (d) are performed multiple times.

4. The method of claim 3, wherein steps (c) and (d) are performed from about 10 to about 25 times.

5. The method of either of claims 1 or 2, wherein the solution contains a compound that confers viscosity.

6. The method of claim 5, wherein the compound is glycerol or a sugar.

7. The method of claim 6, wherein the compound is glycerol.

8. The method of claim 6, wherein the compound is a sugar.

9. The method of either of claims 1 or 2, wherein the DNA polymerase is a thermostable polymerase.

10. The method of either of claims 1 or 2, wherein synthesis and denaturation are performed at different temperatures.

11. The method of either of claims 1 or 2, further comprising detecting the duplexes.

12. The method of claim 11, wherein the oligonucleotide primers are labeled.

13. The method of claim 12, wherein the label is a fluorescent molecule.

14. The method of claim 12, wherein the label is a tag that is detectable by non-fluorescent spectrometry or potentiometry.

15. The method of claim 14, wherein the detection of the tag is by mass spectrometry, infrared spectrometry, ultraviolet spectrometry, or poteniostatic amperometry.

16. The method of claim 14, wherein the sequence and the tag of the first or second or both oligonucleotide primers is different for each template.

17. The method of claim 16, wherein the amplified nucleic acids are pooled prior to detection.

18. The method of either of claims 1 or 2, wherein the array is on a solid substrate comprising a silicon wafer or borosilicate slide.

19. The method of claim 18, wherein the templates are covalently attached to the solid substrate.

20. The method of claim 19, wherein the attachment is through a polyethylene imine linkage.

22. The method of either of claims 1 or 2, wherein the template is uniformly applied to the entire array prior to mixing.

23. The method of either of claims 1 or 2, wherein the template is applied individually to each discrete area on the substrate.

24. The method of claim 23, wherein the applying is performed using spring probes.

25. The method of either of claims 1 or 2, wherein an apparatus is used to control the dew point.

2. A method of amplifying nucleic acid molecules from a template in a chamber, comprising:

(a) mixing single-stranded nucleic acid templates on a solid substrate with a solution comprising a first oligonucleotide primer that hybridizes to the templates, a second oligonucleotide primer that hybridizes to a complementary strand of the template, and a DNA polymerase, wherein the mixing occurs in discrete areas on the substrate, and wherein the solution remains in the discrete areas;

(b) synthesizing a complementary strand to the template to form a duplex;

(c) denaturing the duplex; and

(d) synthesizing complementary strands to the template and the complementary strand of the template, therefrom amplifying nucleic acid molecules;

wherein the solution is in contact with the atmosphere in the chamber, and dew point is maintained during said mixing, synthesizing, and denaturing, thereby preventing evaporation of the solution.

21. The method of claim 2, wherein the oligonucleotide primer pairs each have a different sequence.

26. A method of synthesizing a nucleic acid molecule from a template, comprising:

(a) mixing single-stranded nucleic acid templates on a solid substrate with a solution comprising an oligonucleotide primer that hybridizes to the templates and a DNA polymerase, wherein the mixing occurs in a discrete area of an array, and wherein the solution remains in discrete areas; and

(b) synthesizing a complementary strand to the template to form a duplex,

wherein mixing and synthesis are performed at dew point,

wherein dew point is achieved by an apparatus, comprising;

a container capable of being pressurized;

a heating device;

a means for generating pressure; and

a means for generating saturated steam;

wherein the heating device, pressure generating means, and steam genearting means are controllable.

29. The method of claim 26 wherein step (b) is performed multiple times.

30. The method of claim 26 wherein the solution contains a compound that increases the viscosity of the solution.

31. The method of claim 30 wherein the compound is glycerol or a sugar.

32. The method of claim 30 wherein the array is located on a substantially flat surface of a substrate.

33. The method of claim 32 wherein the substrate is glass.

27. A method of detecting a single base alteration in a nucleic acid molecule, comprising:

(a) mixing single-stranded nucleic acid molecules on a solid substrate with a solution comprising a first and a second oligonucleotides that hybridize to the nucleic acid molecules and a DNA ligase, wherein the mixing occurs in a discrete area of an array, and wherein the solution remains in the discrete areas; and

(b) detecting a ligation product;

wherein the first and second oligonucleotides will not ligate when there is a single base alteration at the junction base on the nucleic acid molecule, p1 mixing is performed at dew point,

wherein dew point is achieved by an apparatus, comprising;

a container capable of being pressurized;

a heating device;

a means for generating pressure; and

a means for generating saturated steam;

wherein the heating device, pressure generating means, and steam generating means are controllable.

34. The method of claim 27 wherein the solution contains a compound that increases the viscosity of the solution.

35. The method of claim 34 wherein the compound is glycerol or a sugar.

36. The method of claim 27 wherein the nucleic acid molecules form an array on the substrate, and the array is located on a substantially flat surface of the substrate.

37. The method of claim 36 wherein the substrate is glass.

28. A method of performing single nucleotide extension assay, comprising:

(a) mixing oligonucleotides on a solid substrate with a solution comprising single-stranded nucleic acid molecules that hybridize to the oligonucleotides, a single nucleotide, and a DNA polymerase, wherein the mixing occurs in discrete areas of the substrate, and wherein the solution remains in discrete areas; and

(b) detecting an extension product of the oligonucleotide;

wherein the oligonucleotide will be extended only when the single nucleotide is complementary to the nucleotide adjacent to the hybridized oligonucleotide,

wherein mixing is performed at dew point,

wherein dew point is achieved by an apparatus, comprising;

a container capable of being pressurized;

a heating device;

a means for generating pressure; and

a means for generating saturated steam;

wherein the heating device, pressure generating means, and steam generating means are controllable.

38. The method of claim 28 wherein the solution contains a compound that increases the viscosity of the solution.

39. The method of claim 38 wherein the compound is glycerol or a sugar.

40. The method of claim 28 wherein the oligonucleotides form an array on the substrate, and the array is located on a substantially flat surface of the substrate.

41. The method of claim 40 wherein the substrate is glass.