Mass spectrometry to assess DNA sequence polymorphisms

• US 5,869,242 A
• Filed: 09/18/1995
• Issued: 02/09/1999
• Est. Priority Date: 09/18/1995
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Myriad Genetics, Inc.
• Inventors: Kamb, Alexander
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US08/529,879
• Time in Patent Office: 1,240 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 435/91.53, 435/172.3, 435/320.1, 436/94, 436/173, 436/174, 935/76, 935/77, 250/282
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: H01J 49/04 Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

A method for determining the presence of polymorphisms, including mutations, in nucleic acids by using mass spectrometry is presented. The method requires amplification of the nucleic acid region to be analyzed followed by analysis by mass spectrometry and comparison of the obtained spectrum with spectra obtained from wild-type sequences and/or sequences known to contain the polymorphism. Differences between the spectra, either the appearance or disappearance of one or more peaks indicating a change in mass or a change in the height of one or more peaks indicating a change in the amount of nucleic acid of a specific mass, indicate the presence of a polymorphism. Variations of the method involve digestion of the amplified nucleic acid, e.g., by using restriction enzymes, nucleases or chemical methods, prior to analysis by mass spectrometry. The method can be applied to any type of nucleic acid including genomic DNA, CDNA and RNA. The method is especially well suited for performing routine genetic screening on a large scale for mutations known to be associated with a disease. The method is also appropriate for determining the presence of polymorphisms for other purposes, e.g., for genotyping or screening for mutations in a positional cloning project. A preferred approach is to amplify then digest the nucleic acid and then to analyze it via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a neodymium-garnet laser and a 3-hydroxypicolinic acid matrix.

Claims

1. A method to analyze for a polymorphism or a mutation in a gene or a portion of said gene encoded by a nucleic acid by

a) denaturing said nucleic acid or a portion of said nucleic acid to produce a denatured nucleic acid,

b) performing mass spectrometry on said denatured nucleic acid to obtain a mass spectrum,

c) comparing the obtained mass spectrum with reference mass spectra obtained of the nucleic acid in its wild-type, polymorphic, or mutated state, and

d) determining whether the obtained mass spectrum matches a reference spectrum for either the wild-type nucleic acid or the nucleic acid having said polymorphism or mutation, wherein a match with said wild-type nucleic acid indicates that said gene is wild-type and a match with said nucleic acid having said polymorphism or mutation indicates that said gene has said polymorphism or mutation, a match being indicated by identity of peak locations (representing mass) and relative peak heights (representing quantity), with the proviso that said method does not comprise sequencing said nucleic acid.

2. The method according to claim 1 wherein a portion of said nucleic acid is analyzed.

3. A method to analyze for a polymorphism or a mutation in a portion of a gene encoded by a nucleic acid by

a) denaturing said nucleic acid encoding said portion of said gene to produce a denatured nucleic acid,

b) amplifying said denatured nucleic acid to produce an amplified nucleic acid,

c) performing mass spectrometry on said amplified nucleic acid to obtain a mass spectrum,

d) comparing the obtained mass aspect with reference mass spectra obtained of the nucleic acid in its wild-type, polymorphic, or mutated state, and

e) determining whether the obtained mass spectrum matches a reference spectrum for either the wild-type nucleic acid or the nucleic acid having said polymorphism or mutation, wherein a match with said wild-type nucleic acid indicates that said gene is wild-type and a match with said nucleic acid having said polymorphism or mutation indicates that said gene has said polymorphism or mutation, a match being indicated by identity of peak locations (representing mass) and relative peak heights (representing quantity), with the proviso that said method does not comprise sequencing said nucleic acid.

4. The method according to claim 3 wherein said amplified nucleic acid is digested prior to denaturation and analysis.

5. The method according to claim 4 wherein digestion is performed using one or more restriction endonucleases.

6. The method according to claim 4 wherein digestion is performed using a deoxyribonuclease.

7. The method according to claim 4 wherein digestion is performed using chemical cleavage.

8. The method according to claim 4 wherein following digestion the digested nucleic acid is denatured and a single-stranded nucleic acid fragment is purified and analyzed.

9. The method according to claim 5 wherein following digestion the digested nucleic acid is denatured and a single-stranded nucleic acid fragment is purified and analyzed.

10. The method according to claim 3 wherein the amplified nucleic acid is denatured and a single-stranded nucleic acid fragment is purified and analyzed.

11. The method according to claim 3 wherein said amplification produces an RNA fragment.

12. A method to analyze for a polymorphism or mutation in a portion of a gene encoded by a nucleic acid by

a) cloning said nucleic acid into a vector containing one or more RNA promoters,

b) synthesizing RNA using said vector with said nucleic acid as a template,

c) performing mass spectrometry on said RNA to obtain a mass spectrum,

d) comparing the obtained mass spectrum with reference mass spectra obtained of the RNA in its wild-type, polymorphic, or mutated state, and

e) determining whether the obtained mass spectrum matches a reference spectrum for either the wild-type RNA or the RNA having said polymorphism or mutation, wherein a match with the wild-type RNA indicates that said gene is wild-type and a match with said RNA having said polymorphism or mutation indicates that said gene has said polymorphism or mutation, a match being indicated by identity of peak locations (representing mass) and relative peak heights (representing quantity), with the proviso that said method does not comprise sequencing said RNA.