Methods and apparatus for performing submicroliter reactions with nucleic acids or proteins

• US 7,138,254 B2
• Filed: 02/07/2003
• Issued: 11/21/2006
• Est. Priority Date: 08/02/1999
• Status: Expired due to Term

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: GE Healthcare UK Limited (Danaher Corporation)
• Inventors: Li, Jeng-Thun, Salas-Solano, Oscar, Jovanovich, Stevan Bogdan
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/361,481
• Publication Number:
  US 20030224395A1
• Time in Patent Office: 1,383 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/00585 Parallel processes
• 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 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 3/0241 Drop counters; Drop formers making arrays for combinatorial libraries B01J19/0046; automation of dispensing for analysis G01N35/10
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• 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 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

Methods for preparing nanoscale reactions using nucleic acids or proteins are presented. Nucleic acids are captured saturably, yet reversibly, on the internal surface of the reaction chamber, typically a capillary. Excess nucleic acid is removed and the reaction is performed directly within the capillary. Proteins are captured specifically and saturably on the modified inner surface of the reaction chamber, typically a capillary. Excess protein is removed and the reaction is performed directly within the capillary. Devices for effecting the methods of the invention and a system designed advantageously to utilize the methods for high throughput reactions involving nucleic acids or proteins are also provided.

Claims

1. A method of performing an enzymatic reaction in a capillary tube using a normalized quantity of a nucleic acid, comprising:

saturably binding a nucleic acid from an excess thereof directly on an inner surface of said capillary tube by contacting said inner surface with a solution comprising said nucleic acid and a chaotropic agent for a time sufficient for the nucleic acid to become saturably bound to said inner surface;

removing unbound excess of the nucleic acid therefore generating a normalized quantity of said nucleic acid;

introducing an enzymatic reaction mixture into said capillary tube having said normalized quantity of said nucleic acid;

wherein said reaction mixture comprises an oligonucleotide primer, a DNA polymerase, dNPTs, and at least one dideoxynucleotide triphosphate (ddNTP),

performing said enzymatic reaction in said capillary tube using said normalized quantity of said nucleic acid.

2. The method of claim 1, further comprising subjecting said enzymatic reaction mixture to at least one thermal cycle.

3. The method of claim 1, further comprising, after said step of removing said excess of the nucleic acid, a step of washing said inner surface of said capillary tube.

4. The method of claim 3, further comprising, after said step of washing said inner surface of said capillary tube, a step of drying said inner surface of said capillary tube.

5. The method of claim 1, wherein said enzymatic reaction mixture is introduced into said capillary tube by capillary action.

6. The method of claim 1, further comprising, after said step of performing said enzymatic reaction, a step of expelling the product of said enzymatic reaction.

7. The method of claim 1, further comprising, after said step of performing said enzymatic reaction, a step of removing unincorporated dideoxynucleotide triphosphates (ddNTPs).

8. The method of claim 7, wherein said unincorporated ddNTPs are removed by contacting the product of said enzymatic reaction with gel filtration media.

9. The method of claim 1, further comprising, after said step of performing said enzymatic reaction, a step of inactivating unincorporated dideoxynucleotide triphosphates (ddNTPs).

10. The method of claim 9, wherein said unincorporated ddNTPs are inactivated by treating the product of said enzymatic reaction with calf intestinal alkaline phosphatase (CIAP).

11. The method of claim 1, wherein the dideoxynucleotide triphosphates (ddNTPs) included in said enzymatic reaction mixture are selected from the group consisting of ddATP only;

ddCTP only;

ddGTP only;

ddTTP only;

ddATP and ddCTP;

ddATP and ddGTP;

ddATP and ddTTP;

ddCTP and ddGTP;

ddCTP and ddTTP;

ddGTP and ddTTP;

ddATP, ddCTP, and ddGTP;

ddATP, ddCTP, and ddTTP;

ddATP, ddGTP, and ddTTP;

ddCTP, ddGTP, and ddTTP; and

ddATP, ddGTP, ddCTP, and ddTTP.

12. The method of claim 1, wherein said dideoxynucleotide triphosphate (ddNTP) is conjugated to a fluorophore.

13. The method of claim 12, wherein said fluorophore is base-specific.

14. The method of claim 12, wherein said fluorophore is selected from the group consisting of:

fluorescein, 5-carboxy-fluorescein, 6-carboxy-rhodnmine, N,N,N′

,N′

-tetramethyl-5-crboxyrhodamine, 5-carboxy-x-rhodamine, rhodamine 110, rhodamine-6-G, tetramethyl rhodamine and rhodamine X.

15. The method of claim 12, wherein said fluorophore is an energy-transfer fluorophore.

16. The method of claim 1, further comprising analyzing a product of said enzymatic reaction to determine the identity of a ddNTP incorporated at the 3′

-end of the primer.

17. The method of claim 16, wherein said step of analyzing a product of said enzymatic reaction to determine the identity of a ddNTP present in said nucleic acid is effected using a technique or an apparatus selected from the group consisting of gel electrophoresis, capillary electrophoresis, mass spectroscopy, MALDI mass spectroscopy, SELDI mass spectroscopy, fluorescence emission detection, scanning confocal laser-induced fluorescence detection, fluorescence polarization (FP) and analytical microchip analysis.

18. The method of claim 16, further comprising inferring the identity of said ddNTP incorporated at the 3′

-end of said primer from the emission spectrum of a fluorophore conjugated to said ddNTP.

19. The method of claim 18, further comprising inferring the identity of a nucleotide present in said nucleic acid from the identity of said ddNTP incorporated at the 3′

-end of said primer.

20. The method of claim 19, wherein the identity of said nucleotide present in said nucleic acid defines a single nucleotide polymorphism (SNP) in said nucleic acid.

21. The method of claim 20, wherein the identity of said nucleotide is stored as data in a computer data structure.

22. The method of claim 21, wherein said computer data structure is embodied in a computer readable medium.

23. The method of claim 1, wherein said DNA polymerase is thermostable.

24. The method of claim 1, wherein said DNA polymerase is a DNA-dependent DNA polymerase.

25. The method of claim 1, wherein said DNA polymerase is an RNA-dependent DNA polymerase.

26. The method of claim 1, wherein said nucleic acid is selected from the group consisting of:

DNA, double stranded DNA, single stranded DNA, DNA produced by polymerase chain reaction, DNA produced by reverse transcription reaction, DNA isolated from an eukaryotic cell, DNA isolated from a prokaryotic cell, DNA isolated from an archaea cell, DNA isolated from a fungal cell, DNA isolated from a plant cell, DNA isolated from a virus, DNA isolated from a bacteriophage, genomic DNA, plasmid DNA, episomal DNA, RNA, messenger RNA, double stranded RNA, single stranded RNA, RNA isolated from an eukaryotic cell, RNA isolated from a prokaryotic cell, RNA isolated from an archaea cell, RNA isolated from a fungal cell, RINA isolated from a plant cell, RNA isolated from a virus, DNA-RNA hybrid, nucleic acid obtained from frozen glycerol stocks of bacteria and nucleic acid obtained from bacterial colonies grown on solid growth media.

27. The method of claim 1, wherein said nucleic acid is DNA;

and further comprising the step of preparing said DNA by polymerase chain reaction (PCR).

28. The method of claim 27, wherein said nucleic acid is genomic DNA.

29. The method of claim 27, further comprising, after said step of preparing said DNA by PCR, a step of removing unincorporated PCR primer using a DNase which can cut single stranded DNA.

30. The method of claim 27, further comprising, after said step of preparing said DNA by PCR, a step of removing unincorporated dNTPs using a phosphatase.

31. The method of claim 27, further comprising, after said step of preparing said DNA by PCR, a step of treating said DNA with Exonuclease 1 (ExoI) and shrimp alkaline phosphatase (SAP).

32. The method of claim 1, further comprising, after said step of saturably binding said nucleic acid from the excess thereof directly on said inner surface of said capillary tube and removing said unbound excess of the nucleic acid therefrom, a step of removing unincorporated PCR primer and dNTPs by washing said inner surface of said capillary.

33. The method of claim 1, wherein said enzymatic reaction is performed in a reaction volume of about 10–

5000 nanoliters.

34. The method of claim 1, wherein said capillary tube is present in a spatially addressable array of capillary tubes.

35. The method of claim 34, wherein said spatially addressable array of capillary tubes is an array having a number of capillaries selected from the group consisting of:

2, 4, 8, 12, 16, 24, 32, 48, 64, 96, 128, 192, 288, 384, 480, 576, 672, 768, 864, 960 and 1536 capillaries.