DNA diagnostics based on mass spectrometry

• US 6,221,605 B1
• Filed: 02/15/2000
• Issued: 04/24/2001
• Est. Priority Date: 03/17/1995
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Köster, Hubert
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/504,245
• Time in Patent Office: 434 Days
• Field of Search: 435/6, 435/91.2, 435/91.1
• US Class Current: 435/5
• 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.

Claims

1. A process for detecting a target nucleic acid sequence present in a biological sample, comprising the steps of:

a) obtaining a target nucleic acid sequence from a biological sample;

b) replicating the target nucleic acid sequence, thereby producing a replicated molecule;

c) specifically digesting the replicated nucleic acid molecule using at least one appropriate nuclease, thereby producing digested fragments;

d) immobilizing the digested fragments onto a solid support containing complementary capture nucleic acid sequences to produce immobilized fragments; and

e) analyzing the immobilized fragments by mass spectrometry, wherein the determination of the molecular weight of the immobilized fragments provide information on the target nucleic acid sequence.

13. A process of claim 1, wherein the solid support is selected from the group consisting of beads, flat surfaces, pins, combs and wafers.

2. A process for detecting one or more target nucleic acids in a biological sample, comprising:

a) digesting one or more nucleic acids using at least one nuclease, thereby producing digested fragments;

b) analyzing the digested fragments by mass spectrometry, whereby detection of the target nucleic acid by mass spectrometry indicates the presence of the target nucleic acid sequence in the biological sample.

3. The process claim 2, further comprising the step of amplifying one or more target nucleic acids.

4. The method of claim 2, wherein the digested fragments are conditioned prior to mass spectrometric analysis.

5. The method of claim 2, wherein the nuclease is a restriction endonuclease.

6. The process of claim 2, wherein identification of a target nucleic acid in the sample provides a genetic diagnosis, detects chromosomal aneuploidy, detects a genetic predisposition to a disease or condition, or detects or identifies infection by a pathogen.

7. The process of claim 2, wherein a plurality of nucleic acids from the sample are immobilized on a solid support prior to mass spectrometric analysis.

8. The method of claim 7, wherein the nucleic acids are arranged in an array and each spot on the array is subjected to mass spectrometric analysis.

9. The process of claim 7, wherein immobilization is effected by a bond cleavable by a pyrophosphatase.

10. The process of claim 2, wherein a nucleic acid comprising the target sequence has been contacted with an alkylating agent prior to mass spectrometric analysis.

11. The process of claim 2, wherein a nucleic acid comprising the target sequence includes one or more of the following:

nucleotides that reduce sensitivity for depurination, RNA building blocks, phosphorothioate groups, nucleic acid mimetics and protein nucleic acid (PNA).

12. The process of claim 11, wherein the target nucleic acid includes an alkylated phosphorothioate group.