Methods and apparatus for template capture and normalization for submicroliter reaction

• US 6,927,045 B2
• Filed: 09/30/2002
• Issued: 08/09/2005
• Est. Priority Date: 08/02/1999
• Status: Expired due to Fees

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Amersham Biosciences SV Corp (Thermo Fisher Scientific Incorporated)
• Inventors: Hadd, Andy, Jovanovich, Stevan
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/262,476
• Publication Number:
  US 20030032052A1
• Time in Patent Office: 1,044 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/18.3, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• 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 2400/0487 fluid pressure, pneumatics
• 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 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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
• CPC Class Codes: Y10S 435/809 : Incubators or racks or holders for culture plates or containers
• CPC Class Codes: Y10T 436/113332 : with conveyance of sample along a test line in a container or rack
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

Methods for preparing nanoscale reactions using nucleic acids 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. Alternatively, the saturably bound nucleic acid is eluted, dispensing a metered amount of nucleic acid for subsequent reaction in a separate chamber. Devices for effecting the methods of the invention and a system designed advantageously to utilize the methods for high throughput nucleic acid sequencing reactions are also provided.

Claims

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

introducing an enzymatic reaction mixture into a capillary tube, said tube having a normalized quantity of DNA,

wherein said reaction mixture comprises an oligonucleotide primer, a DNA polymerase, and deoxynucleotide triphosphates (dNTPs),

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

said excess of DNA having been removed therefrom; and

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

2. The method of claim 1, further comprising the step of size selecting the DNA prior to said step of introducing said enzymatic reaction mixture into said capillary tube.

3. The method of claim 2, wherein said step of size-selecting is effected by the exclusion of genomic DNA from the bore of said capillary.

4. The method of claim 1, wherein said reaction mixture further comprises dideoxynucleotide triphosphates.

5. The method of claim 4, wherein said dideoxynucleotide triphosphates are conjugated to a fluorophore.

6. The method of claim 5, wherein said fluorophore is selected from among the group consisting of:

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

,N′

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

7. The method of claim 5, wherein said fluorophore is an energy transfer fluorophore.

8. The method of claim 1, wherein said primer is complementary to a plurality of contiguous nucleotides in said DNA.

9. The method of claim 8, wherein said primer is conjugated to a fluorophore.

10. The method of claim 9, wherein said fluorophore is selected from among the group consisting of:

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

,N′

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

11. The method of claim 9, wherein said fluorophore is an energy transfer fluorophore.

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

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

14. The method of claim 1, wherein said DNA is selected from among the group consisting of eukaryotic DNA, prokaryotic DNA, animal DNA, plant DNA, fungal DNA, archea DNA, mammalian DNA, human DNA, virus DNA, bacteriophage DNA, lambda phage DNA, M13 phage DNA, bacterial DNA, E. coli DNA, genomic DNA, plasmid DNA, episomal DNA, DNA produced by polymerase chain reaction and DNA to be spotted onto a substrate to create a microarray.

15. The method of claim 1, further comprising analyzing a product of said enzymatic reaction to determine the sequence of said DNA.

16. The method of claim 15, wherein a product of said enzymatic reaction is purified prior to said step of analyzing said product to determine the sequence of said DNA.

17. The method of claim 16, wherein said purification is effected using a technique selected from among the group consisting of alcohol precipitation, size exclusion chromatography, size exclusion chromatography using Sephadex®

and treatment with combination of enzymes.

18. The method of claim 16, wherein said step of analyzing a product of said enzymatic reaction to determine the sequence of said DNA is effected using a technique selected from among the group consisting of electrophoresis, capillary electrophoresis, gel electrophoresis, mass spectroscopy, MALDI mass spectroscopy, SELDI mass spectroscopy, fluorescence detection, scanning confocal laser-induced fluorescence detection and analytical microchip analysis.

19. The method of claim 16, wherein said step of analyzing a product of said enzymatic reaction to determine the sequence of said DNA is effected by single base extension reactions.