Base specific cleavage of methylation-specific amplification products in combination with mass analysis
Inventors
Ehrich, Mathias • VAN DEN BOOM, Dirk Johannes
Assignees
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Publication Number
US-9249456-B2
Publication Date
2016-02-02
Expiration Date
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.
Core Innovation
The invention relates to determining the methylation state of one or more nucleotide loci in a target nucleic acid molecule by using methylation-state dependent nucleotide modification, amplification, base-specific cleavage, and mass spectrometry. The method generates mass signals from a fragmented target nucleic acid molecule in which a reagent modifies a nucleotide according to whether the nucleotide is methylated, and the target nucleic acid is then contacted with a base-specific cleavage reagent that cleaves the target nucleic acid into fragments.
A central aspect of the invention is comparing fragment mass patterns to corresponding reference mass signals derived from base-specific cleavage or simulated cleavage of a reference nucleic acid molecule. The references correspond to methylated and/or unmethylated loci, and the fragment mass pattern includes determining the presence of a fragment, the absence of a fragment, and the mass of a fragment relative to a reference.
The methylation state is determined based on the fragment mass patterns obtained from these comparisons. The comparison is grounded in fragments of at least three nucleotides in length, enabling locus-level determination from fragmentation and fragment mass patterns. The disclosed approach also includes configurations involving specific base-specific cleavage reagents and methylation-dependent nucleotide modification reagents such as bisulfite.
Claims Coverage
The independent claim defines a method to determine methylation state at one or more nucleotide loci by generating mass-spectrometry signals from fragmented, methylation-modified and amplified target nucleic acids, comparing resulting fragment mass patterns to reference patterns for methylated and/or unmethylated loci, and determining methylation state based on the comparison. Three inventive features are captured below.
Mass-spectrometry signals from methylation-modified amplified target nucleic acid with base-specific cleavage
Generating by mass spectrometry mass signals of a fragmented target nucleic acid molecule, wherein the target nucleic acid molecule is treated with a reagent that modifies a nucleotide according to whether the nucleotide is methylated, amplified, and contacted with a base-specific cleavage reagent that cleaves the target nucleic acid into fragments.
Fragment mass pattern comparison to methylated and unmethylated reference mass signals
Comparing the mass signals of fragments of at least three nucleotides in length of the target nucleic acid molecule to 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 loci are methylated and/or unmethylated, to determine fragment mass patterns including determining the presence of a fragment, the absence of a fragment, and the mass of a fragment relative to a reference.
Determining methylation state based on fragment mass patterns
Determining the methylation state of the one or more nucleotide loci in the target nucleic acid molecule based on the fragment mass patterns determined by comparing mass signals to the reference mass signals.
Across the independent claim, the core inventive coverage lies in producing fragment mass signals from methylation-modified and amplified target nucleic acids using base-specific cleavage, deriving locus-specific fragment mass patterns by comparing to methylated and unmethylated reference or simulated cleavage patterns, and determining methylation state based on fragment presence, absence, and relative mass.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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