DNA sequencing by mass spectrometry via exonuclease degradation

• US 5,872,003 A
• Filed: 05/30/1995
• Issued: 02/16/1999
• Est. Priority Date: 03/19/1993
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Koster, Hubert
• Primary Examiner(s): Redding, David A.
• Application Number:
  US08/453,499
• Time in Patent Office: 1,358 Days
• Field of Search: 435/286.1, 435/287.2, 435/288.7, 435/287.3, 935/77, 935/86, 935/88
• US Class Current: 435/283.1
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/2429 of arylalkanols
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 35/1067 for transfer to or from containers having different spacing

Abstract

Methods for determining the sequence of nucleic acids by cleaving the nucleic acid unilaterally from a first end with an exonuclease activity to sequentially release individual nucleotides, identifying each of the sequentially release nucleotides by mass spectrometry, and determining the sequence of the nucleic acid from the identified nucleotides are disclosed. The method is amenable to multiplexing for simulataneously determining more than one nuleic acid sequence.

Claims

1. A system for exonuclease-mediated mass spectrometric sequencing comprising:

(i) a reactor and capillary tube means for performing and containing exonuclease cleavage reactions of the target nucleic acids, the cleavage reaction forming a train of sequentially released individual nucleotides;

(ii) mass spectrometric means for detecting the train of individual nucleotides released by the exonuclease cleavage of the target nucleic acid; and

(iii) transfer means for transferring the train of individual nucleotides from the reactor means to the mass spectrometric means.

2. The system of claim 1, wherein the transfer means further comprises a moving belt.

3. The system according to claim 2, in which the moving belt comprises an endless moving belt driven by a stepping motor and held under tension by spring-loaded pulleys.

4. A system of claim 2, which is additionally comprised of a sample application area for applying a sample of the individual nucleotides released by the exonuclease cleavage of the target nucleic acids to a portion of the moving belt.

5. A system of claim 2, which is additionally comprised of a camera with optics for viewing the sample application area.

6. A system of claim 2, which is additionally comprised of a source for ionization/desorption capable of moving across the surface of the belt in a direction perpendicular to the motion of the moving belt.

7. The system according to claim 6, in which the ionization/desorption source is a laser.

8. A system of claim 2, which is additionally comprised of a differential vacuum pumping stages for generating a vacuum over a portion of the moving belt.

9. A system of claim 2, which is additionally comprised of a heating device to facilitate removal of sample residue from the moving belt.

10. The system according to claim 9, further comprising a washing station to wash the moving belt with a cleaning solution prior to application of the sample, wherein the heating device facilitates drying the belt alter rinsing with the cleaning solution.

11. The system of claim 9, wherein the heating device is a microwave.

12. The system according to claim 1, wherein the reactor means further comprises a reactor with a cooling/heating mantle for controlling the temperature of the exonuclease cleavage reaction.

13. The system according to claim 12, wherein the reactor means further comprises at least one secondary reactor for mass-modifying the individual nucleotides released by the exonuclease cleavage of the target nucleic acids.

14. The system according to claim 13, wherein the secondary reactor comprises immobilized alkaline phosphatase.

15. The system according to claim 1, further comprising a plurality of reactor means for parallel sequencing of a plurality of nucleic acids.

16. The system of claim 1, further comprising a microprocessor for processing the detected train of released nucleotides and determining the sequence of the target nucleic acids.

17. A system of claim 1, which is additionally comprised of at least one reagent flask for providing at least one reagent to the reactor and at least one pumping device for pumping the reagents and reactants to and from the reactor.

18. A system of claim 1, which is additionally comprised of at least one heating/cooling coil for controlling the temperature of reagents provided to the reactor.

19. A system of claim 1, which is additionally comprised of a means for immobilizing one of the target nucleic acids or the exonuclease in the reactor.

20. A system of claim 1, wherein exonuclease is immobilized in the reactor.

21. A system of claim 1, wherein the target nucleic acids are immobilized in the reactor.