DETERMINATION OF METHYLATION STATUS OF POLYNUCLEOTIDES

• US 20110091882A1
• Filed: 10/01/2010
• Published: 04/21/2011
• Est. Priority Date: 10/02/2009
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Laffler, Thomas G., Granados, Edward N.
• Application Number:
  US12/896,574
• Publication Number:
  US 20110091882A1
• Time in Patent Office: Days
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention provides compositions and methods for detecting the methylation status of a nucleic acid. In particular, the present invention provides a mass spectrometry-based method of determining DNA methylation status without sequencing.

Claims

1. A method of determining the methylation status of a nucleic acid, the method comprising:

(a) reacting said nucleic acid with bisulfate;

(b) amplifying one or more segments said nucleic acid using at least one purified oligonucleotide primer pair to produce an amplification product; and

(c) determining the mass and/or base composition of said amplification product, thereby determining said methylation status of said nucleic acid.

2. The method of claim 1, wherein said nucleic acid comprises DNA.

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

4. The method of claim 1, wherein said nucleic acid comprises a DNA promoter.

5. The method of claim 1, wherein bisulfate reacts with unmethylated cytosine residues, converting them to uracil residues.

6. The method of claim 1, wherein bisulfate does not react with methylated cytosine residues, leaving them as 5-methylcytosine.

7. The method of claim 1, wherein amplifying one or more segments said nucleic acid comprises PCR.

8. The method of claim 1, wherein (c) comprises detecting a molecular mass of said amplification product.

9. The method of claim 1, wherein (c) comprises determining a base composition of said amplification product, wherein said base composition identifies the number of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof in said amplification product, whereby said base composition indicates the methylation status of said nucleic acid.

10. The method of claim 9, comprising comparing said base composition of said amplification product to calculated or measured base compositions of amplification products present in a database with the proviso that sequencing of said amplification product is not used to indicate the methylation status, wherein a match between the determined base composition and the calculated or measured base composition in said database indicates methylation status.

11. The method of claim 9, comprising comparing said base composition of said amplification product to the base composition of a control nucleic acid with the proviso that sequencing of said amplification product is not used to indicate the methylation status, wherein differences in mass between the determined base composition and control base composition indicates methylation status.

12. The method of claim 1, further comprising an initial step of isolating said nucleic acid from a subject or sample.

13. A system for determining the methylation status of a nucleic acid, the system comprising:

(a) instrumentation for calculating a molecular mass of a nucleic acid molecule; and

(b) a database comprising masses or base compositions of known bisulfate converted nucleic acid molecules.

14. The system of claim 13, further comprising (c) reagents for bisulfate treatment of a nucleic acid molecule.