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

摘要

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.

主权项

1. A method for determining the methylation state of one or more nucleotide loci in a target nucleic acid molecule, which comprises:
(a) generating by mass spectrometry mass signals of a fragmented target nucleic acid molecule, wherein the target nucleic acid molecule has been (i) treated with a reagent that modifies a nucleotide according to whether the nucleotide is methylated, (ii) amplified and (iii) contacted with a base-specific cleavage reagent that cleaves the target nucleic acid into fragments, (b) comparing the mass signals of fragments of at least three nucleotides in length of the target nucleic acid molecule to the corresponding reference mass signals of fragments of at least three nucleotides in length derived from base-specific cleavage or simulated cleavage of a reference nucleic acid molecule in which each of the one or more nucleotide loci are methylated and/or the corresponding reference mass signals fragments of at least three nucleotides in length derived from base-specific cleavage or simulated cleavage of a reference nucleic acid molecule in which each of the one or more nucleotide loci are unmethylated to determine fragment mass patterns, wherein a fragment mass pattern includes determining: (i) the presence of a fragment, (ii) the absence of a fragment, and (iii) the mass of a fragment relative to a reference, and c) determining the methylation state of the one or more nucleotide loci in the target nucleic acid molecule based on the fragment mass patterns determined in (b).