DNA diagnostics based on mass spectrometry

• US 7,198,893 B1
• Filed: 10/10/2000
• Issued: 04/03/2007
• Est. Priority Date: 11/06/1996
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Braun, Andreas, Hillenkamp, Franz, van den Boom, Dirk, Koster, Hubert, Jurinke, Christian, Ruppert, Andreas, Little, Daniel P.
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US09/686,148
• Time in Patent Office: 2,366 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/25.3, 536/24.4
• US Class Current: 435/6.16
• 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 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00504 Pins
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• 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 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 : separated from the reactor apparatus
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 3/0268 : using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07F 9/2408 : of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/2429 : of arylalkanols
• 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 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 : Allele-specific amplification
• CPC Class Codes: C12Q 1/6865 : Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6872 : involving mass spectrometry
• CPC Class Codes: C12Q 2523/10 : Characterised by chemical treatment
• CPC Class Codes: C12Q 2525/197 : incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2531/113 : PCR
• 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 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/627 : being a mass spectrometer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/1037 : Using surface tension, e.g. pins or wires
• CPC Class Codes: G01N 2035/1069 : by adjusting the spacing between multiple probes of a single transferring head
• CPC Class Codes: G01N 35/1067 : for transfer to or from containers having different spacing
• CPC Class Codes: G01N 35/1074 : arranged in a two-dimensional array
• CPC Class Codes: H01J 49/00 : Particle spectrometers or separator tubes

Abstract

Fast and highly accurate mass spectrometry-based processes for detecting a particular nucleic acid sequence in a biological sample are provided. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

Claims

1. A process for determining the order of base specifically terminated nucleic acid fragments of a target nucleic acid molecule, comprising the steps of:

a. obtaining a nucleic acid molecule, comprising the target nucleic acid sequence and, at one end, a tag;

b. generating base specifically terminated nucleic acid fragments from the target nucleic acid; and

c. analyzing the fragments by a mass spectrometry format, thereby determining the order of the base specifically terminated nucleic acid fragments in the target nucleic acid molecule.

2. A process of claim 1, wherein in step b), a nuclease is contacted with the target nucleic acid to generate the base specifically terminated nucleic acid fragments.

3. A process of claim 2, wherein the nuclease is a restriction enzyme that can recognize and cleave at least one restriction site in the target nucleic acid.

4. A process of claim 2, wherein the target nucleic acid is a deoxyribonucleic acid and the nuclease is a deoxyribonuclease.

5. A process of claim 2, wherein the target nucleic acid is a ribonucleic acid and the nuclease is a ribonuclease.

6. A process of claim 5, wherein the ribonuclease is selected from the group consisting of:

the G-specific T₁ ribonuclease, the A-specific U₂ ribonuclease, the A/U specific PhyM ribonuclease, the U/C specific ribonuclease A, the C-specific chicken liver ribonuclease and crisavitin.

7. A process of claim 1, wherein in step b), the base specifically terminated nucleic acid fragments are generated by performance of a combined amplification and base-specific termination reaction, wherein a first polymerase facilitates amplification and a second polymerase facilitates termination.

8. A process of claim 7, wherein the combined amplification and base-specific termination reaction is performed using a first polymerase, which has a relatively low affinity towards at least one chain terminating nucleotide, and a second polymerase, which has a relatively high affinity towards at least one chain terminating nucleotide.

9. A process of claim 8, wherein the first and second polymerases are thermostable DNA polymerases.

10. A process of claim 9, wherein the thermostable DNA polymerases are selected from the group consisting of:

Taq DNA polymerase, AmpliTaq FS DNA polymerase, Deep Vent (exo-) DNA polymerase, Vent DNA polymerase, Vent (exo⁻

) DNA polymerase, Vent DNA polymerase, Vent (exo⁻

) DNA polymerase, Deep Vent DNA polymerase, Thermo Sequenase, exo(-) Pseudococcusfiuriosus (Pfu) DNA polymerase, AmpliTaq, Ultman, 9 degree Nm, Tth, Hot Tub, Pyrococcus furiosus (Pfu) and Pyrococcus woesei (Pwo) DNA polymerase.

11. A process of claim 1, wherein the base specifically terminated nucleic acid fragments generated in step b) include mass modified nucleotides.

12. A process of claim 1, wherein the tag comprises a 3′

tag.

13. A process of claim 1, wherein the tag comprises a 5′

tag.

14. A process of claim 12 or 13, wherein the tag is a non-natural tag.

15. A process of claim 14, wherein the non-natural tag is selected from the group consisting of:

an affinity tag and a mass marker.

16. A process of claim 15, wherein the affinity tag facilitates immobilization of the nucleic acid to a solid support.

17. A process of claim 16, wherein the affinity tag is biotin or a nucleic acid sequence that binds to a capture nucleic acid sequence that is bound to a solid support.