Apparatus for DNA sequencing

• US 6,296,810 B1
• Filed: 06/18/1997
• Issued: 10/02/2001
• Est. Priority Date: 02/01/1993
• Status: Expired due to Fees

• Current Assignee: Amersham Biosciences Corp. (General Electric Company)
• Original Assignee: Praelux, Inc. (General Electric Company)
• Inventors: Ulmer, Kevin M.
• Primary Examiner(s): ALEXANDER, LYLE
• Application Number:
  US08/877,606
• Time in Patent Office: 1,567 Days
• Field of Search: 356/344, 250/461.2, 436/94, 436/86, 436/96, 422/82.05, 422/82-7-, 422/64-65, 435/6
• US Class Current: 422/82.07
• 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 device for determining a sequence of bases in nucleic acid comprising:

a light source for irradiating a local area having a base therein with excitation light, said local area being on a flat substrate holding a sequence of said bases of nucleic acid;

fluorescence detecting means for detecting fluorescence generated from said base present in said local area on said flat substrate and for identifying a kind of said base based on a wavelength distribution of said fluorescence and a lifetime of said fluorescence;

moving means for moving said substrate; and

means for controlling said moving means so as to relatively move said local area on said flat substrate along said sequence of bases.

2. A device according to claim 1, wherein

said fluorescence detecting means is disposed at a position which is out of an optical path of said excitation light irradiated by said light source and of a reflected light from said surface of said substrate.

3. A device according to claim 2, wherein said light source includes a laser device.

4. A device according to claim 1, comprises cooling means for cooling said local area on said surface of the substrate.