Methods and apparatus for template capture and normalization for submicroliter reaction

• US 6,489,112 B1
• Filed: 08/02/2000
• Issued: 12/03/2002
• Est. Priority Date: 08/02/1999
• Status: Expired due to Fees

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Dynamics Inc. (General Electric Company)
• Inventors: Jovanovich, Stevan, Hadd, Andy
• Primary Examiner(s): Whibenant, Ethan C.
• Assistant Examiner(s): Lu, Frank W
• Application Number:
  US09/632,094
• Time in Patent Office: 853 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.1, 435/287.2, 422/68.1, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.11
• 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 obtaining substantially the same quantity of nucleic acid from a first and a second sample, comprising:

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

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

wherein said inner surfaces of said first and second capillary tubes are capable of saturably binding substantially the same quantity of nucleic acid from each of said first and second samples, respectively.

2. The method of claim 1, wherein the quantity of nucleic acid saturably bound to the inner surfaces of said first and second capillary tubes differs by less than about 10%.

3. The method of claim 1, wherein said binding steps are effected substantially contemporaneously.

4. The method of claim 1, wherein said second capillary tube is the same capillary tube as said first capillary tube, and wherein said binding steps are effected iteratively.

5. The method of claim 1 further comprising, prior to said binding steps, the step of size-selecting a nucleic acid to be saturably bound.

6. The method of claim 1 further comprising, after said binding steps, the step of using the nucleic acid of either of said first or second capillary tubes in an enzymatic reaction.

7. The method of claim 1 wherein the saturably bound nucleic acid of either of said first or second capillary tubes is DNA.

8. The method of claim 7 further comprising, after said binding steps, the step of using the DNA of either of said first or second capillary tubes in an enzymatic reaction.

9. The method of claim 8 wherein said enzymatic reaction is a DNA sequencing reaction.

10. The method of claim 1, wherein either of said first or second capillary tubes comprises glass.

11. The method of claim 1, wherein said capillary tubes are present in an array.

12. The method of claim 11, wherein said array comprises at least 8 capillary tubes.

13. The method of claim 11, wherein said array comprises at least 16 capillary tubes.

14. The method of claim 11, wherein said array comprises at least 96 capillary tubes.

15. The method of claim 1 wherein said chaotropic agent is selected from the group consisting of:

urea, sodium perchlorate, potassium perchlorate, sodium bromide, potassium bromide, sodium iodide, potassium iodide, sodium thiocyanate, potassium thiocyanate, guanidine thiocyanate, sodium isothiocyanate, potassium isothiocyanate, guanidine hydrochloride, guanidine isothiocyanate, lithium chloride, sodium trichloroacetate, dimethylsulfoxide, tetra-amine halides, tetraethylamine chloride, and potassium trichloroacetate.

16. The method of claim 1 further comprising the step of removing the solution, wherein said removing step occurs after said contacting step.

17. The method of claim 16 further comprising the step of washing the inner surface of either of said first or second capillary tubes, wherein said washing step occurs after said removing step.

18. The method of claim 17 further comprising the step of drying the inner surface of either of said first or second capillary tubes, wherein said drying step occurs after said washing step.

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

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

said nucleic acid 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 a nucleic acid and a chaotropic agent for a time sufficient for the nucleic acid to have become saturably bound to said inner surface; and

said excess of nucleic acid having been removed therefrom.

20. The method of claim 19 further comprising the step of introducing into said capillary tube an enzymatic reaction mixture after said excess of nucleic acid has been removed therefrom.