Nucleic acid diagnostics based on mass spectrometry or mass separation and base specific cleavage

• US 6,994,960 B1
• Filed: 05/28/1998
• Issued: 02/07/2006
• Est. Priority Date: 05/28/1997
• Status: Expired due to Fees

• Current Assignee: The Walter & Eliza Hall Institute Of Medical Research
• Original Assignee: The Walter & Eliza Hall Institute Of Medical Research
• Inventors: Simpson, Richard, Reid, Gavin, Foote, Simon, Elso, Colleen
• Primary Examiner(s): MYERS, CARLA J
• Assistant Examiner(s): Chakrabarti, Arun
• Application Number:
  US09/424,629
• Time in Patent Office: 2,812 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.21, 435/91.3, 435/196, 536/24.3, 536/23.1, 935/6, 935/77, 436/518, 436/94, 250/282, 250/288
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

A method of detecting a mutation or a difference of one or more nucleotides between a nucleic acid molecule to be tested and a reference nucleic acid molecule, said method comprising subjecting the test nucleic acid molecule to base specific cleavage to generate oligonucleotide fragments, separating the resulting oligonucleotide fragments based on mass by MALDI-ATOF MS and/or other equivalent procedure to produce a fingerprint of then oligonucleotide fragments comprising one or more peaks wherein a peak represents the mass of each fragment and identifying an altered peak relative to a reference nucleic acid molecule subjected to the same procedure wherein the presence of an altered peak is indicative of a difference of one or more nucleotides in said tested nucleic acid molecule.

Claims

1. A method of detecting a difference of one or more nucleotides between a nucleic acid molecule to be tested and a reference nucleic acid molecule wherein the location of said one or more nucleotide that are different between the nucleic acid molecule to be tested and the reference nucleic acid molecule is unknown , said method comprising subjecting the test nucleic acid molecule to single-base-specific cleavage to generate oligonucleotide fragments, separating the resulting oligonucleotide fragments based on mass by MALDI-TOF MS to produce a fingerprint of the oligonucleotide fragments comprising one or more peaks wherein a peak represents the mass of each fragment and identifying an altered peak relative to said reference nucleic acid molecule subjected to the same procedure wherein the presence of an altered peak is indicative of a difference of one or more nucleotides in said tested nucleic acid molecule relative to said reference nucleic acid molecule.

2. A method according to claim 1 wherein the nucleic acid molecule to be tested is amplified by a polymerase chain reaction (PCR) prior to base specific cleavage.

3. A method according to claim 1 wherein the base specific cleavage results in oligonucleotide fragments of from about 2 bases to about 1000 bases.

4. A method according to claim 3 wherein the base specific cleavage results in oligonucleotide fragments of from about 3 bases to about 500 bases.

5. A method according to claim 4 wherein the base specific cleavage results in oligonucleotide fragments of from about 4 bases to about 100 bases.

6. A method according to claim 1 wherein the base specific cleavage is uracil specific cleavage.

7. A method according to claim 6 wherein the uracil specific cleavage is mediated by uracil-N-glycosylase.

8. A method according to claim 1 further comprising subjecting fragmentation products to further separation (PSD) to generate a spectrum from decay dependent on the nucleotide sequence of the oligonucleotide.

9. A method according to claim 8 wherein the further separation of fragmentation products is by post source decay (PSD).

10. A method for identifying or locating a mutation in one or more bases in a target nucleic acid molecule wherein the location of said mutation within the target nucleic acid molecule is unknown, comprising subjecting the target nucleic acid molecule to single-base-specific cleavage to generate oligonucleotide fragments, separating the resulting olignucleotide fragments based on mass by MALDI-TOF MS to produce a fingerprint of the oligonucleotide fragments comprising one or more peaks wherein a peak represents the mass of each fragment, and identifying an altered peak relative to said reference nucleic acid molecule subjected to the same procedure, wherein the presence of an altered peak is indicative of a mutation in one or more bases in said target nucleic acid molecule relative to said reference nucleic acid molecule.

11. A method according to claim 10 wherein the nucleic acid to be tested is amplified by a polymerase chain reaction (PCR) prior to base specific cleavage.

12. A method according to claim 10 wherein the base specific cleavage results in oligonucleotide fragments of from about 2 bases to about 1000 bases.

13. A method according to claim 12 wherein the base specific cleavage results in oligonucleotide fragments of from about 3 bases to about 500 bases.

14. A method according to claim 13 wherein the base specific cleavage results in oligonucleotide fragments of from about 4 bases to about 100 bases.

15. A method according to claim 10 wherein the base specific cleavage is uracil specific cleavage.

16. A method according to claim 15 wherein the uracil specific cleavage is mediated by uracil-N-glycosylase.

17. A method according to claim 10 further comprising subjecting the oligonucleotide fragments to further separation to generate a spectrum from decay dependent on the nucleotide sequence of the oligonucleotide.

18. A method according to claim 17 wherein the further separation of fragmentation products is by post source decay (PSD).

19. A method of detecting a difference of one or more nucleotides between a nucleic acid molecule acid molecule to be tested and a reference nucleic acid molecule wherein the location of said one or more nucleotides that are different between the nucleic acid molecule to be tested and the reference nucleic acid molecule is unknown, said method comprising subjecting the test molecule to single-base-specific cleavage to generate oligonucleotide fragments, separating the resulting oligonucleotide fragments based on mass by MALDI-TOF MS to produce a fingerprint of the oligonucleotide fragments comprising one or more peaks wherein a peak represents the mass of each fragment, and identifying an altered peak relative to said reference nucleic acid molecule subjected to the same procedure, wherein the presence of an altered peak is indicative of said difference of one or more nucleotdies in said tested nucleic acid molecule relative to said reference nucleic acid molecule, and wherein the difference does not result in a change of a cleavage site.

20. A method for identifying or locating a mutation in one or more bases in a target nucleic acid molecule wherein the mutation does not result in a charge of a cleavage site by a restriction enzyme and wherein the location of said mutation within the target nucleic acid molecule is unknown, comprising subjecting the target nucleic acid molecule to single-base-specific cleavage to generate oligonucleotide fragments, separating the resulting oligonucleotides fragments based on mass by MALDI-TOF MS to produce a fingerprint of the oligonucleotide fragments comprising one or more peaks wherein a peak represents the mass of each fragment, and identifying an altered peak relative to a reference nucleic acid molecule subjected to the same procedure, wherein the presence of an altered peak is indicative of a mutation in one or more bases in said target nucleic acid molecule relative to said reference nucleic acid molecule, and wherein the difference does not result in a change of a cleavage site by a restriction enzyme.