DNA diagnostics based on mass spectrometry

• US 6,602,662 B1
• Filed: 11/28/2000
• Issued: 08/05/2003
• Est. Priority Date: 03/17/1995
• 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, Little, Daniel P., Köster, Hubert
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/724,877
• Time in Patent Office: 980 Days
• Field of Search: 435/6, 435/91.1
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: G01N 35/1067 for transfer to or from containers having different spacing
• CPC Class Codes: H01J 49/00 : Particle spectrometers or separator tubes
• 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

Fast and highly accurate mass spectrometry-based processes for detecting particular nucleic acid molecules and sequences in the molecules are provided. Depending upon the sequence to be detected, the processes, for example, can be used to diagnose a genetic disease or a chromosomal abnormality, a predisposition to a disease or condition, or infection by a pathogen, or for determining identity or heredity. One aspect provides a process for determining whether a target nucleotide is present in a nucleic acid molecule including hybridizing a nucleic acid molecule with a primer oligonucleotide; contacting the hybridized primer with deoxyribonucleoside triphosphates, chain terminating nucleotides and a DNA polymerase, whereby the hybridized primer is extended until a chain terminating nucleotide is incorporated, producing an extended primer, and determining the molecular mass of the extended primer, thereby determining whether the target nucleotide is present in a nucleic acid molecule.

Claims

1. A process for determining whether a target nucleotide is present in a nucleic acid molecule, comprising:

a) hybridizing a nucleic acid molecule with a primer oligonucleotide that is complementary to a sequence that is adjacent to a region suspected of containing the target nucleotide, thereby producing a hybridized primer;

b) contacting the hybridized primer with

i) deoxyribonucleoside triphosphates,

ii) chain terminating nucleotides selected from the group consisting of dideoxyribonucleoside triphosphates and 3′

-deoxynucleoside triphosphates, and

iii) a DNA polymerase, whereby the hybridized primer is extended until a chain terminating nucleotide is incorporated, thereby producing an extended primer; and

c) determining the molecular mass of the extended primer, thereby determining whether the target nucleotide is present in a nucleic acid molecule.

4. The process of claim 1, wherein the presence of a target nucleotide is indicative of the presence of a mutation.

6. The process of claim 1, wherein the primer is immobilized.

2. A process for detecting the presence of a target nucleotide in a nucleic acid molecule, comprising:

a) hybridizing a detector oligonucleotide with a sample containing a nucleic acid molecule suspected of containing the target nucleotide;

b) removing unhybridized detector oligonucleotide;

c) extending the detector oligonucleotide; and

d) determining, by mass spectrometry, the molecular mass of a molecule that comprises the detector oligonucleotide, thereby detecting the presence of a target nucleotide or sequence thereof in the sample.

3. The process of claim 2, wherein the target nucleotide or sequence thereof comprises a mutation.

5. The process of claim 2, wherein the presence of a target nucleotide is indicative of the presence of a mutation.

7. The process of claim 2, wherein the detector is immobilized.