Compositions and methods for preserving DNA methylation
Inventors
Markowitz, Sanford D. • Moinova, Helen • Wurtman, David
Assignees
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Publication Number
US-12553089-B2
Publication Date
2026-02-17
Expiration Date
Abstract
The disclosure provides methods for storage solutions for preserving DNA methylation patterns over a period of time. The disclosure also provides for methods of using methylated DNA stored in such storage solutions.
Core Innovation
The invention relates to preserving DNA cytosine methylation patterns in biological samples using a methanol-containing aqueous storage solution. The storage solution comprises peroxide-free methanol, or methanol at a peroxide level less than or equal to 0.001%, and is free of metal ions.
The invention addresses preserving methylation patterns such that a threshold proportion of the original methylation patterns present prior to storage is preserved after storage. In particular, at least 60% of the methylation patterns present in the methylated DNA sequence prior to storage are preserved when stored for at least 24 hours in the aqueous storage solution under the claimed conditions.
The invention further describes maintaining methylation signals while avoiding time-dependent increases and spurious methylation artifacts observed with comparative preservatives. The document includes experimental comparisons of methanol-based solutions against other preservatives such as CytoLyt®, PreservCyt®, and neutral buffered formalin, focusing on preservation of true methylation patterns and assay reproducibility.
Claims Coverage
The partial content provides four independent inventive features that share the same core concept: preserving methylation patterns after treating human esophageal cells with a methanol-containing aqueous storage solution. Each independent claim specifies quantitative preservation of methylation patterns and constrains the storage solution chemistry.
Aqueous methanol storage solution preserving methylation patterns at room temperature
A method of preserving methylation patterns of a methylated DNA sequence in human esophageal cells by treating the cells with an aqueous storage solution at room temperature, storing for at least 24 hours, extracting DNA after storing, wherein the aqueous storage solution comprises from 40% to less than 100% methanol and the methanol is peroxide free or at a level less than or equal to 0.001%.
Metal-ion-free methanol aqueous storage solution with preservation threshold
The method wherein the aqueous storage solution is free of metal ions, the extracted DNA comprises a methylated DNA sequence with methylation patterns, and at least 60% of the methylation patterns present prior to storing are preserved when stored for at least 24 hours in the aqueous storage solution.
Ambient temperature methanol storage with preservation threshold for methylation patterns
A method of preserving methylation patterns of a methylated DNA sequence by treating human esophageal cells with an aqueous storage solution at room temperature, storing the cells for at least 24 hours at ambient temperature, extracting DNA after storing, wherein the aqueous storage solution comprises from 40% to less than 100% methanol and is free of metal ions.
Preservation of methylation patterns over ambient temperature range
The method wherein the extracted DNA comprises a methylated DNA sequence with methylation patterns and at least 60% of the methylation patterns present prior to storing are preserved when stored for at least 24 hours at ambient temperature ranging between -30° C. to 50° C.
Across the independent claims, the claimed inventive concept is preservation of DNA methylation patterns after treating human esophageal cells with a metal-ion-free aqueous storage solution containing 40% to less than 100% methanol with peroxide-free or low-peroxide methanol, coupled to a quantitative preservation requirement of at least 60% after at least 24 hours under the specified temperature conditions.
Stated Advantages
Preserves methylation patterns such that at least 60% of the methylation patterns present prior to storage are preserved after at least 24 hours in the claimed aqueous storage solution.
Maintains methylation signals over prolonged times, with reported avoidance of time-dependent increases and spurious methylation artifacts compared with comparative preservatives.
Supports room-temperature storage while maintaining methylation pattern preservation for the claimed thresholds.
Documented Applications
Preservation of methylation patterns of a methylated DNA sequence in human esophageal cells for subsequent DNA extraction.
Use of preserved methylation patterns in methylation assays described in the document, including assay workflows involving bisulfite conversion and methylation assays.
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