MULTIPLEX METHYLATION-SPECIFIC AMPLIFICATION SYSTEMS AND METHODS

• US 20140274735A1
• Filed: 03/13/2014
• Published: 09/18/2014
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Inventors: Granados, Edward
• Granted Patent: US 9,701,999 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

The present invention relates to systems and methods for performing multiplex amplification reactions. In particular, the present invention relates to multiplex methylation-specific amplification systems and methods.

Claims

1. A method, comprising:

a) treating a target nucleic acid with a methylation-specific detection reagent;

b) simultaneously amplifying a plurality of nucleic acid segments within said target nucleic acid to generate a plurality of amplicons; and

c) detecting said plurality of amplicons.

2. The method of claim 1, wherein said target is a promoter region of a gene.

3. The method of claim 1, wherein said methylation specific reagent is bisulfite.

4. The method of claim 1, wherein said simultaneous amplifying is carried out in a single reaction vessel.

5. The method of claim 1, wherein said nucleic acid target is GC-rich.

6. The method of claim 5, wherein said nucleic acid target comprises CpG islands.

7. The method of claim 3, wherein bisulfite reacts with unmethylated cytosine residues, converting them to uracil residues.

8. The method of claim 3, wherein bisulfite does not react with methylated cytosine residues, leaving them as 5-methylcytosine.

9. The method of claim 1, wherein said amplification is PCR.

10. The method of claim 1, wherein said detecting comprises contacting said amplicons with a plurality of nucleic acid probes.

11. The method of claim 10, wherein said nucleic acid probes comprise a detectable label.

12. The method of claim 11, wherein said plurality of nucleic acid probes comprise the same label.

13. The method of claim 4, wherein said amplification and detection comprises real time PCR amplification and detection.

14. The method of claim 1, wherein said detecting comprises a nucleic acid detection technique selected from the group consisting of mass spectroscopy, sequencing, and hybridization.

15. The method of claim 1, wherein said plurality of amplicons comprises at least 5 amplicons.

16. The method of claim 1, wherein said plurality of amplicons comprises at least 10 amplicons.

17. A method, comprising:

a) treating a target nucleic acid with a methylation-specific detection reagent;

b) simultaneously amplifying a plurality of nucleic acid segments within said target nucleic acid to generate a plurality of amplicons; and

c) contacting said amplicons with a plurality of nucleic acid probes to detect said plurality of amplicons.

18. A kit, comprising:

a) a plurality of primer pairs for amplification of a target nucleic acid;

b) a plurality of nucleic acid probes that specifically bind to amplicons amplified by said plurality of primer pairs; and

c) a methylation specific detection reagent.

19. The kit of claim 18, wherein said probes comprise a detectable label.

20. The kit of claim 19, wherein said probes comprise the same label.