Instrument and method for the sequencing of genome

• US 5,288,644 A
• Filed: 11/13/1992
• Issued: 02/22/1994
• Est. Priority Date: 04/04/1990
• Status: Expired due to Term

• Current Assignee: Rockefeller University
• Original Assignee: Rockefeller University
• Inventors: Beavis, Ronald C., Chait, Brian T.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): REDDING, DAVID A
• Application Number:
  US07/957,688
• Time in Patent Office: 466 Days
• Field of Search: 435/6, 436/89, 436/94, 436/173, 436/807, 250/282, 536/27, 536/28, 536/29, 935/77, 935/87
• US Class Current: 436/94
• 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 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00628 Ionic
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• 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: H01J 49/0413 for automated handling
• CPC Class Codes: H01J 49/164 Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] sample holders H01J49/0418
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

A method for DNA sequencing, and particularly for sequencing of the entire human genome. Different base-specific reactions are utilized to use different sets of DNA fragments from a piece of DNA of unknown sequence. Each of the different sets of DNA fragments has a common origin and terminates at a particular base along the unknown sequence. The molecular weight of the DNA fragments in each of the different sets is detected by a matrix assisted laser absorption mass spectrometer to determine the sequence of the different bases in the DNA. The methods and apparatus of the present invention provide a relatively simple and low cost technique which may be automated to sequence thousands of gene bases per hour, and eliminates the tedious and time consuming gel electrophoresis separation technique conventionally used to determine the masses of DNA fragments.

Claims

1. A method of DNA sequencing of the genome by detecting the mass weights of DNA fragments comprising the steps of:

(a) producing a piece of DNA of unknown sequence;

(b) performing four different base-specific reactions on the piece of DNA to produce four different DNA sets containing DNA fragments each DNA set having a common origin and terminating at a particular base along the unknown sequence;

(c) selecting a solid matrix having a strong absorption band at the wavelength of light produced by a laser and placing one of the DNA sets on the matrix, the strong absorption band of the matrix having a longer wavelength than the absorption bands for the DNA set;

(d) without separation of the DNA fragments by electrophoresis or other separation methods and without labeling the DNA fragments with radioactive, fluorescent or other labeling means, striking the DNA fragments on the solid matrix within one of the DNA sets with a series of laser pulses from the laser to desorb ions of the DNA fragments and produce ionized DNA fragments;

(e) detecting the mass weights of the ionized DNA fragments by a time of flight mass spectrometer; and

(f) repeating steps (d) and (e) for the other DNA sets to determine the sequence of bases in the DNA.

2. The method as defined in claim 1, wherein each different set of DNA fragments are mixed with the selected solid matrix.

3. The method as defined in claim 1, wherein the surface of the solid matrix has a plurality of discrete spots formed thereon, each discrete spot containing one of the different sets of DNA fragments;

step (e) including applying a vacuum and an electric field within the mass spectrometer; and

the step of detecting the mass weights includes detecting the molecular weight of the DNA fragments contained in each spot.

4. The method as defined in claim 3, wherein said plurality of discrete spots on the surface of the solid matrix are each at a fixed location with respect to a reference point on the surface.

5. The method as defined in claim 1, wherein the step of detecting the mass weights further comprises:

determining the absolute mass difference between the detected molecular weight of weight of a peak of one of the sets of DNA fragments compared to a peak of another of the sets of DNA fragments; and

correcting the sequence of the bases in the DNA in response to the determined absolute mass differences.

6. A method of DNA sequencing of the genome, comprising the steps of:

(a) producing a piece of DNA of unknown sequence;

(b) performing at least four different reactions on the piece of DNA to produce at least four different sets containing DNA fragments each having a common origin and terminating at a particular base along the unknown sequence;

(c) striking the DNA fragments within one of the different sets with a series of laser pulses to desorb ions of the DNA fragments;

(d) detecting the mass weights of the ionized fragments within the one set by a time of flight mass spectrometer; and

(e) repeating steps (c) and (d) for other of the different sets of DNA fragments to determine the sequence of bases in the DNA.

7. The method as defined in claim 6, wherein the surface of the solid matrix has a plurality of discrete spots formed thereon, each discrete spot containing one of the different sets of DNA fragments;

step (e) including applying a vacuum and an electric field within the mass spectrometer; and

the step of detecting the mass weights includes detecting the molecular weight of the DNA fragments contained in each spot.

8. The method as defined in claim 7, wherein said plurality of discrete spots on the surface of the solid matrix are each at a fixed location with respect to a reference point on the surface.