Methods and apparatus for DNA sequencing

• US 5,674,743 A
• Filed: 06/05/1995
• Issued: 10/07/1997
• Est. Priority Date: 02/01/1993
• Status: Expired due to Fees

• Current Assignee: Amersham Biosciences Corp. (General Electric Company)
• Original Assignee: SEQ Limited
• Inventors: Ulmer, Kevin M.
• Primary Examiner(s): Redding, David A.
• Application Number:
  US08/463,831
• Time in Patent Office: 855 Days
• Field of Search: 435/287.2, 435/288.7, 422/82.08, 436/94, 436/172
• US Class Current: 435/287.2
• 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 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0663 Stretching or orienting elongated molecules or particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/1838 using fluid heat transfer medium
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0268 : using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502776 : specially adapted for focusing or laminating flows
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 2521/325 : Single stranded exonuclease
• CPC Class Codes: C12Q 2537/157 : A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/601 : being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 2565/631 : being a biochannel or pore
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/145555 : Hetero-N

Abstract

The present invention provides a method and apparatus for automated DNA sequencing. The method of the invention includes the steps of: a) using a processive exonuclease to cleave from a single DNA strand the next available single nucleotide on the strand; b) transporting the single nucleotide away from the DNA strand; c) incorporating the single nucleotide in a fluorescence-enhancing matrix; d) irradiating the single nucleotide to cause it to fluoresce; e) detecting the fluorescence; f) identifying the single nucleotide by its fluorescence; and g) repeating steps a) to f) indefinitely (e.g., until the DNA strand is fully cleaved or until a desired length of the DNA is sequenced). The apparatus of the invention includes a cleaving station for the extraction of DNA from cells and the separation of single nucleotides from the DNA; a transport system to separate the single nucleotide from the DNA and incorporate the single nucleotide in a fluorescence-enhancing matrix; and a detection station for the irradiation, detection and identification of the single nucleotides. The nucleotides are advantageously detected by irradiating the nucleotides with a laser to stimulate their natural fluorescence, detecting the fluorescence spectrum and matching the detected spectrum with that previously recorded for the four nucleotides in order to identify the specific nucleotide.

Claims

1. A genome sequencer comprising:

means for sequentially separating single nucleotides from a strand of DNA;

means for confining the separated nucleotides in their original sequence in a solid matrix;

means for exciting each separated nucleotide;

means for detecting the spectroscopic emission of each separated nucleotide; and

means for identifying each separated nucleotide according to its spectroscopic emission.

2. The genome sequencer according to claim 1 wherein means for exciting each separated nucleotide includes a laser system.

3. The genome sequencer according to claim 2 wherein the laser system includes a mode-locked laser.

4. The genome sequencer according to claim 3 wherein the mode-locked laser operates in tunable wavelength region from approximately 240 nm to 300 nm.

5. The genome sequencer method according to claim 4 wherein the mode-locked laser operates at a repetition rate of 76 MHz.

6. The genome sequencer according to claim 5 wherein the mode-locked laser operates with a pulse width of approximately one picosecond.

7. The genome sequencer according to claim 1 wherein means for detecting the spectroscopic emission of each separated nucleotide includes means for measuring by single photon counting the fluorescence spectrum of each separated nucleotide.

8. The genome sequencer according to claim 7 wherein means for measuring by single photon counting the fluorescence spectrum of each separated nucleotide includes a monochromator.

9. The genome sequencer according to claim 7 further comprising means for time gating the single photon counting.

10. The genome sequencer according to claim 1 wherein means for detecting the spectroscopic emission of each separated nucleotide includes means for measuring by single photon counting the fluorescence lifetime of each separated nucleotide.

11. The genome sequencer according to claim 10 further comprising means for time correlating the single photon counting.

12. The genome sequencer according to claim 11 wherein means for time correlating includes a time-to-amplitude converter.

13. The genome sequencer according to claim 12 wherein means for time correlating further includes a multi-channel analyzer.

14. The genome sequencer according to claim 1 wherein means for detecting includes a micro-channel plate photomultiplier.

15. The genome sequencer according to claim 1 wherein means for detecting includes a single-photon avalanche diode.

16. The genome sequencer according to claim 1 10 wherein means for detecting includes a streak camera.

17. The genome sequencer according to claim 1 further comprising optical fibers for collecting the spectroscopic emission.

26. The genome sequencer of claim 1 wherein the means for separating includes an optical trap suitable for positioning a microsphere to which said strand of DNA is affixed.

27. The genome sequencer according to claim 1 wherein the means for separating includes means for fixing said separated nucleotides in a solid support.

18. A genome sequencer comprising:

(a) means for sequentially separating single nucleotides from a strand of DNA in the order of the original sequence of said nucleotides in said strand; and

(b) a refrigeration system capable of cooling a solution containing said separated nucleotides to a temperature sufficient to vitrify the solution containing said separated nucleotides.

19. The genome sequencer according to claim 18 wherein the means for separating includes an optical trap suitable for positioning a microsphere to which said strand of DNA is affixed.

20. The genome sequencer according to claim 18 wherein the means for separating includes means for depositing said separated nucleotides on a solid support.

21. The genome sequencer according to claim 18 wherein the means for separating includes (i) an optical trap suitable for positioning a microsphere to which said strand of DNA is affixed;

and (ii) means for depositing said separated nucleotides on a transparent support in a thin continuous liquid film or as discrete droplets;

said sequencer further comprising a radiation source for irradiating said nucleotides so as to cause said nucleotides to fluoresce, and means for moving said support from a first position where said nucleotides are cooled by said refrigeration system to a second position where said nucleotides are irradiated by said radiation source.

22. The genome sequencer according to claim 21 further comprising means for detecting fluorescence of said nucleotides.

23. The genome sequencer according to claim 18 further comprising a radiation source for irradiating the nucleotides so as to cause said nucleotides to fluoresce.

24. The genome sequencer according to claim 23 further comprising means for detecting fluorescence of said nucleotides.

25. A genome sequencer comprising:

(a) a sheath, laminar flow microchannel;

(b) means for regulating flow through the microchannel so as to achieve hydrodynamic focusing; and

(c) a refrigeration system capable of imposing a temperature gradient over the microchannel, sufficient to vitrify an aqueous solution within said microchannel.