Multiplex methylation-specific amplification systems and methods

• US 9,701,999 B2
• Filed: 03/13/2014
• Issued: 07/11/2017
• Est. Priority Date: 03/14/2013
• Status: Expired due to Fees

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Inventors: Granados, Edward
• Primary Examiner(s): Kim, Young J
• Application Number:
  US14/208,862
• Publication Number:
  US 20140274735A1
• Time in Patent Office: 1,216 Days
• Field of Search: None
• 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 specific target nucleic acid with a methylation-specific detection reagent, wherein the specific target nucleic acid is a single gene or untranslated region of a single gene;

b) simultaneously amplifying a plurality of different nucleic acid segments within said specific target nucleic acid to generate a plurality of different amplicons wherein said simultaneous amplifying is carried out in a single reaction vessel; and

c) detecting two or more amplicons of said plurality of different amplicons comprising said different nucleic acids segments within said specific target nucleic acid wherein said detecting comprises contacting said plurality of different amplicons comprising said different nucleic acids segments within said specific target nucleic acid with a plurality of different nucleic acid probes wherein said plurality of different nucleic acid probes comprise a detectable label that is the same label in each of said plurality of different nucleic acid probes.

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

5. The method of claim 4, wherein said nucleic acid target comprises CpG islands.

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

7. The method of claim 1, wherein said amplification and detection comprises real time PCR amplification and detection.

8. The method of claim 1, wherein said detecting comprises detecting hybridization of at least two detectably labeled probes of said plurality of different nucleic acid probes to at least two amplicons of the plurality of different amplicons.

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

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

11. A method, comprising:

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

b) simultaneously amplifying a plurality of different nucleic acid segments within said specific target nucleic acid to generate a plurality of different amplicons comprising said different nucleic acids segments within said specific target nucleic acid wherein said simultaneous amplifying is carried out in a single reaction vessel; and

c) contacting said amplicons with a plurality of different nucleic acid probes to detect said plurality of amplicons comprising said different nucleic acids segments within said specific target nucleic acid wherein said plurality of different nucleic acid probes comprise a detectable label that is the same label in each of said plurality of different nucleic acid probes.