Base specific cleavage of methylation-specific amplification products in combination with mass analysis

• US 20050272070A1
• Filed: 03/24/2005
• Published: 12/08/2005
• Est. Priority Date: 03/26/2004
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Van Den Boom, Dirk Johannes, Ehrich, Mathias
• Granted Patent: US 9,249,456 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

Methods, combinations and kits are provided for identifying the methylation state of a target nucleic acid molecule, the methylation state of a nucleotide locus in a target nucleic acid molecule, or for identifying the locus of one or more methylated or unmethylated nucleotides in a target nucleic acid molecule. Methylation state identification is performed by treating a methylated target nucleic acid molecule with a reagent that modifies one or more nucleotides in the target nucleic acid molecule as a function of the methylation state of the target nucleic acid molecule, methylation specifically amplifying treated target nucleic acid molecule, fragmenting amplified products, and detecting one or more fragments to thereby identify the methylation state of a target nucleic acid molecule, the methylation state of a nucleotide locus in a target nucleic acid molecule, or the locus of one or more methylated or unmethylated nucleotides in a target nucleic acid molecule.

Claims

1. A method of identifying a methylated nucleic acid molecule, comprising:

(a) treating a target nucleic acid molecule with a reagent that modifies unmethylated cytosine to produce uracil;

(b) specifically amplifying the treated target nucleic acid molecule with a primer whose target nucleic acid binding region contains one or more guanine nucleotides;

(c) base specifically cleaving the amplified products; and

(d) detecting the cleaved products, wherein the presence of two or more fragments indicates that the target nucleic acid molecule contains one or more methylated cytosines.

2. A method of discovering methylation correlated with a disease, disease outcome, or outcome of a treatment regimen, comprising the steps of:

(a) identifying methylated or unmethylated nucleotides, in accordance with the method of any of claim 1, in one or more nucleic acid molecules from one or more samples collected from one or more subjects having a known disease, disease outcome, or outcome of a treatment regimen;

(b) identifying methylated or unmethylated nucleotides, in accordance with the method of any of claim 1, in one or more nucleic acid molecules from one or more samples collected from one or more normal subjects; and

(c) identifying the differently methylated or unmethylated nucleotides between the one or more nucleic acid molecules of step (a) and the one or more nucleic acid molecules of step (b);

whereby the differently methylated or unmethylated nucleotides identify methylation correlated with a disease, disease outcome, or outcome of a treatment regimen.

3. The method of claim 1, wherein the reagent for modifying a selected nucleotide is selected from the group consisting of methylmethane sulfonate, ethylmethane sulfonate, diethylsulfate, nitrosoguanidine (N-methyl-N′

-nitro-N-nitrosoguanidine), nitrous acid, di-(2-chloroethyl)sulfide, di-(2-chloroethyl)methylamine, 2-aminopurine, t-bromouracil, hydroxylamine, sodium bisulfite, hydrazine, formic acid, sodium nitrite, and 5-methylcytosine DNA glycosylase.

4. A method of distinguishing between a false positive methylation specific amplification and a true methylation specific amplification, comprising the steps of:

(a) treating a target nucleic acid molecule with a reagent that modifies an unmethylated selected nucleotide to produce a different nucleotide;

(b) contacting the treated target nucleic acid molecule with a methylation state specific primer complementary to a first target nucleic acid region containing one or more of the selected nucleotides;

(c) treating the contacted target nucleic acid molecule under nucleic acid synthesis conditions;

(d) treating the synthesized products under base specific cleavage conditions; and

(e) detecting the mass of the cleaved products, wherein;

a change in mass of one or more cleaved products compared to a reference mass indicates that a nucleotide locus in a second region in a target is methylated, wherein the second region does not overlap with the first region, whereby presence of one or more methylated loci in the second region confirms true methylation specific amplification.