Method and apparatus for performing large numbers of reactions using array assembly with releasable primers

• US 6,632,641 B1
• Filed: 10/10/2000
• Issued: 10/14/2003
• Est. Priority Date: 10/08/1999
• Status: Expired due to Fees

• Current Assignee: Metrigenix Incorporated
• Original Assignee: Metrigenix Incorporated
• Inventors: Berninger, Mark, Chatelain, Francois, Brennan, Thomas M.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/686,597
• Time in Patent Office: 1,099 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24-- 2
• US Class Current: 506/9
• 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 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• 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 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/515 : characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

The present invention relates to a method and apparatus for performing a large number of reactions using array assembly. In particular, the present invention features a method and apparatus for performing a large number of chemical and biological reactions by bringing two arrays into close apposition and allowing reactants on the surfaces of two arrays to come into contact. The present invention is exemplified by performing a large number of polynucleotide amplification reactions using array assembly. In addition, the present invention features a method and apparatus for coupling the amplification of polynucleotides and the detection of sequence variations, expression levels, and functions thereof.

Claims

1. A method for performing a plurality of enzymatic amplification reactions, comprising the steps of:

(a) obtaining a first solid support wherein at least two releasable primers of each reaction are confined to areas on the surface of said first solid support;

(b) obtaining a second solid support with a plurality of wells providing separation of said plurality of reactions;

(c) assembling said first and second solid support;

(d) filling said wells with reactants of said reactions;

(e) releasing said releasable polynucleotides into solution in each well; and

(f) performing said plurality of amplification reactions in parallel.

2. The method according to claim 1 wherein said reactants in step (d) comprise a DNA polymerase, a target nucleic acid, and deoxynucleotides.

10. The method according to any one of the claims 1-2 and 3-9 wherein said areas on the surface of said first solid support are hydrophilic areas.

11. The method according to any one of the claims 1-2 and 3-9 wherein the density of areas on said first solid support is between about 10 to 10,000 per cm².

12. The method according to any one of the claims 1-2 and 3-9 wherein the size of each said area on said first solid support is between about 10⁻

3 to 5 mm².

13. The method according to any one of the claims 1-2 and 3-9 wherein the number of areas on said first solid support is between about 10 to 500,000.

22. The method according to any one of the claims 1-2 and 3-9 wherein said first or second solid support is glass.

23. The method according to claim 1 wherein said releasable primers are released by photolysis in step (e).

24. The method according to claim 1 wherein said releasable primers are released by strand separation in step (e).

25. The method according to claim 1 wherein said releasable primers are released by enzymatic reaction in step (e).

3. A method for performing a plurality of polynucleotide amplification reactions, comprising the steps of:

(a) obtaining first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;

(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;

(c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support; and

(d) releasing said primers;

(e) performing said plurality of polynucleotide amplification reactions.

14. The method according to any one of the claims 3-9 wherein said immobilized moieties are prepared by in situ synthesis.

15. The method according to any one of the claims 3-9 wherein said immobilized moieties are prepared by spotting.

16. The method according to any one of the claims 3-9 wherein said immobilized moieties are prepared using an ink-jet printing like device.

17. The method according to any one of the claims 3-9 wherein each of said plurality of areas on said second solid support is a reaction well.

18. The method according to any one of the claims 3-9 wherein said immobilized moiety on each area of said first solid support in step (a) is a hybridization complex between an immobilized polynucleotide hybridized with a releasable primer for said polynucleotide amplification reactions.

19. The method according to any one of the claims 3-9 wherein said immobilized moiety on each area of said first solid support in step (a) comprises a cleavable site and a releasable primer.

20. The method according to any one of the claims 3-9 wherein said immobilized moiety on each area of said first solid support in step (a) comprises a cleavable site and a releasable primer for said polynucleotide amplification reactions and said cleavable site is cleavable by photolysis.

21. The method according to any one of the claims 3-9 wherein said immobilized moiety on each area of said first solid support comprises a cleavable site and a releasable primer for said polynucleotide amplification reactions and said cleavable site comprises an o-nitrobenzyl linker.

4. A method for performing a plurality of polynucleotide amplification reactions and capturing amplification products, comprising the steps of:

(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;

(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;

(c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;

(d) releasing said primers;

(e) generating amplification products of said polynucleotide amplification reactions; and

(f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either said first or second solid support through hybridization.

5. A method for detecting a plurality of polynucleotide sequence variations, comprising the steps of:

(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;

(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;

(c) assembling said first and second solid support, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;

(d) releasing said primers;

(e) generating amplification products of said polynucleotide amplification reactions;

(f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either said first or second array through hybridization; and

(g) detecting polynucleotide sequence variations by hybridization complexes in step (f).

6. A method for quantitating polynucleotides in a target nucleic acid, comprising the steps of:

(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of finite areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;

(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;

(c) assembling said first and second solid support, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;

(d) releasing said primers;

(e) generating amplification products of said polynucleotide amplification reactions; and

(f) quantitating amplified products.

7. A method for detecting polynucleotide sequence variations in a target nucleic acid, comprising the steps of:

(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;

(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each areas of said second solid support;

(c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;

(d) releasing said primers;

(e) generating amplification products of said polynucleotide amplification reactions; and

(f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either first or second array though hybridization; and

(g) detecting polynucleotide sequence variations by a polynucleotide modifying enzyme.

8. A method for amplifying a target nucleic acid, capturing the amplified product, and detecting a polynucleotide sequence variation in the amplified product, comprising the steps of:

(a) obtaining a first solid support wherein;

(1) the surface of said first solid support comprises a first, second, third, and fourth areas;

(2) a first chemical moiety, comprising a releasable forward primer for said target nucleic acid, is immobilized on said first area;

(3) a second chemical moiety, comprising a releasable reverse primer for said target nucleic acid, is immobilized on said second area;

(4) a first polynucleotide probe, comprising a subsequence complementary to one variant of said polynucleotide variation, is immobilized on said third area said subsequence containing at least one interrogation position complementary to a corresponding nucleotide in said variant; and

(5) a second polynucleotide probe is immobilized to said fourth area, said second polynucleotide probe differing from said first polynucleotide probe by at least one nucleotide;

(b) obtaining a second solid support wherein the surface of said solid support comprises a reaction well and a mixture of reacts comprising a DNA polymerase, said target nucleic acid, and deoxynucleotides are placed within said reaction well;

(c) assembling said first and second solid support, wherein said mixture of reactants are in contact with said first, second, third, and fourth areas on said first solid support;

(d) releasing said releasable forward and reverse primers;

(e) generating the amplified product for said target nucleic acid;

(f) capturing the amplified product by said first or second polynucleotide probe through hybridization;

(g) washing said first solid support;

(h) comparing the relative binding of two probes on said first solid support; and

(i) identifying said polynucleotide variation in the amplified product.

9. A method for amplifying a target nucleic acid, capturing the amplified product, and detecting a polynucleotide sequence variation in the amplified product, comprising the steps of:

(a) obtaining a first solid support wherein;

(1) the surface of said first array comprises a first, second, third, and fourth areas;

(2) a first chemical moiety, comprising a releasable forward primer specific for said region of said target nucleic acid, is immobilized on said first area;

(3) a second chemical moiety, comprising a releasable reverse primer specific for said region of said target nucleic acid, is immobilized on said second area;

(4) a first polynucleotide probe, comprising a subsequence complementary to one variant of said polynucleotide variation, is immobilized on said third area, said subsequence containing at least one interrogation position complementary to a corresponding nucleotide in said variant; and

(5) a second polynucleotide probe is immobilized to said fourth area, said second probe differing from said first probe by at least one nucleotide;

(b) obtaining a second solid support wherein the surface of said solid support comprises a reaction well and a mixture of reactants comprising a DNA polymerase, said target nucleic acid, and deoxynucleotides are placed within said reaction well;

(c) assembling said first and second solid support, wherein said mixture of reactants are in contact with said first, second, third, and fourth areas on said first solid support;

(d) releasing said releasable forward and reverse primers;

(e) generating the amplified product for said target nucleic acid;

(f) capturing the amplified product by said first or second polynucleotide probes through hybridization;

(g) extending said one or more hybridization complexes in step (f);

(h) washing said first solid support; and

(i) identifying said polynucleotide variation using said one or more extended products in step (g).