Methods for detecting methylated nucleotides

• US 7,332,275 B2
• Filed: 10/15/2002
• Issued: 02/19/2008
• Est. Priority Date: 10/13/1999
• Status: Expired due to Term

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Van Den Boom, Dirk, Jurinke, Christian, Braun, Andreas
• Primary Examiner(s): Clow; Lori A.
• Application Number:
  US10/272,665
• Publication Number:
  US 20030180748A1
• Time in Patent Office: 1,953 Days
• Field of Search: 702/19, 702/32, 435/6, 435/183
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

A method for detecting methylated nucleotides within a nucleic acid sample is provided. The method includes the steps of splitting a nucleic acid sample into separate reaction vessels; contacting nucleic acid in one reaction vessel with bisulfite; amplifying the nucleic acid in each reaction vessel; cleaving the nucleic acids in each reaction vessel to produce fragments thereof; and obtaining a mass spectrum of the resulting fragments from one reaction vessel and another mass spectrum of the resulting fragments from another reaction vessel.

Claims

1. A method for detecting methylated nucleotides within a nucleic acid sample, comprising:

splitting a nucleic acid sample into separate reaction vessels;

contacting nucleic acid molecules in one reaction vessel with bisulfite;

amplifying the nucleic acid molecules in each reaction vessel in the presence of dUTP;

cleaving the amplified nucleic acid molecules in each reaction vessel at uracil to produce fragments thereof; and

obtaining a mass spectrum of the resulting fragments from one reaction vessel and another mass spectrum of the resulting fragments from another reaction vessel, whereby;

cytosine methylation is detected by identifying a difference in signals between the mass spectra thereby detecting methylated nucleotides.

2. The method of claim 1, wherein:

the step of amplifying is carried out in the presence of dUTP; and

the step of cleaving further comprises

treating the amplified nucleic acid molecules with a uracil-DNA glycosylase; and

incubating the amplified nucleic acid molecules under conditions effective for cleaving.

3. The method of claim 2, wherein:

the step of incubating said nucleic acid molecules under conditions effective for cleaving further comprises subjecting the nucleic acid molecules to conditions that cleave the phosphate backbone at an abasic site.

4. The method of claim 1, wherein said nucleic acid sample is a pooled sample.

5. The method of claim 1, wherein the mass spectra are obtained using a spectrometric format selected from among Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), Fourier Transform, and combinations thereof.

6. A method for detecting methylated nucleotides within a nucleic acid sample, comprising:

treating nucleic acid molecules with bisulfite to deaminate any unmethylated cytosine to produce uracil;

amplifying the treated nucleic acid molecules in the presence of dUTP;

fragmenting the amplified treated nucleic acid molecules by cleavage at uracil;

obtaining a mass spectrum of the fragmented amplified treated nucleic acid molecules;

obtaining a mass spectrum of fragmented amplified untreated nucleic acid molecules; and

identifying the fragments of amplified treated nucleic acid molecules that contain uracil in place of cytosine by comparing the spectrum from amplified treated fragments with a spectrum resulting from amplified untreated nucleic acid molecules thereby detecting methylated nucleotides.

7. The method of claim 6, wherein the fragmenting step further comprises:

contacting the amplified treated and untreated nucleic acid molecules with a uracil-DNA glycosylase; and

subjecting the nucleic acid molecules to conditions that cleave the phosphate backbone at an abasic site.

8. The method of claim 6, wherein the mass spectra are obtained using a spectrometric format selected from among Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), Fourier Transform, and combinations thereof.

9. A method for detecting methylated nucleotides within a nucleic acid sample, comprising:

treating nucleic acid molecules with bisulfite to deaminate any unmethylated cytosine to produce uracil;

amplifying the treated nucleic acid molecules in the presence of dUTP;

fragmenting the amplified treated nucleic acid molecules by cleavage at uracil;

obtaining a mass spectrum of the fragmented amplified treated nucleic acid molecules;

obtaining a mass spectrum of fragmented amplified untreated nucleic acid molecules; and

comparing the spectrum from amplified treated fragments with the spectrum from amplified untreated nucleic acid molecules;

whereby the presence of at least one methylated cytosine is detected thereby detecting methylated nucleotides.

10. The method of claim 9, wherein the fragmenting step further comprises:

contacting the amplified nucleic acid molecules with a uracil-DNA glycosylase; and

subjecting the nucleic acid molecules to conditions that cleave the phosphate backbone at an abasic site.

11. The method of claim 9, wherein the mass spectra are obtained using a spectrometric format selected from among Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), Fourier Transform, and combinations thereof.

12. A method for identifying the absence, presence or frequency of a polymorphism in a population, wherein said polymorphism is manifested or detected as a difference in post-translational modification, which comprises:

pooling genomic nucleic acid molecules isolated from body tissue or fluid from a plurality of individuals;

treating the nucleic acid molecules with bisulfite to deaminate any unmethylated cytosine to produce uracil;

amplifying the treated nucleic acid molecules;

cleaving the amplified treated nucleic acid molecules at uracil, to produce fragments thereof;

obtaining a mass spectrum of the fragmented amplified treated nucleic acid molecules;

obtaining a mass spectrum of fragmented amplified untreated nucleic acid molecules;

identifying the fragments of amplified treated nucleic acid molecules that contain uracil in place of cytosine by comparing the spectrum from treated amplified treated fragments with a spectrum resulting from amplified untreated nucleic acid molecules and

identifying the absence, presence, or frequency of a polymorphism by determining the allelic frequency of allelic fragments obtained from the pooled genomic nucleic acid molecules.

13. The method of claim 12, wherein the genomic nucleic acid molecules are obtained from healthy subjects.