Detection of DNA methylation
Inventors
Jia, Xiyu • Chimaokereke, Onyinyechi • Nguyen, Lam
Assignees
Publication Number
US-11072817-B2
Publication Date
2021-07-27
Expiration Date
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Abstract
In a first aspect, the invention concerns a method for detecting or quantifying DNA methylation at a locus. In one embodiment, a methylation-sensitive endonuclease is formulated together with a polymerase enzyme in an appropriate reaction mixture such that amplification of DNA occurs in a methylation specific manor. Quantitative DNA amplification at selected loci can be used to determine the level of methylation. Kits and reagents for performing such methods are also provided.
Core Innovation
The invention provides a method for detecting methylation status of a DNA sequence in a DNA sample by contacting the DNA sample with a reaction mixture comprising a methylation-sensitive endonuclease, a DNA polymerase, oligonucleotide primers and a buffer formulated to facilitate activity of the MSE and the DNA polymerase, performing MSE cleavage and DNA polymerization within the same reaction, and detecting DNA amplification wherein DNA amplification is indicative of methylation status of the DNA sequence in the DNA sample.
The background identifies that current methods to analyze DNA methylation include Me-DIP, HPLC, microarrays, mass spectrometry and bisulfite treatment and that bisulfite treatment is labor intensive and complicated to perform; the invention addresses these issues by providing a single reaction system for rapid and accurate DNA methylation quantification that integrates MSE specificity with real-time PCR in one step.
Claims Coverage
One independent claim is presented (claim 1). The claim sets forth a one‑pot premixture and an integrated workflow for MSE cleavage and qPCR, with quantitation of methylation by amplified product.
Premixture composition comprising three specified MSEs and polymerase
A premixture comprising three methylation-sensitive endonucleases (AccII, Hpall, and HpyCH4IV), a thermal stable DNA polymerase, oligonucleotide primers for amplifying the DNA sequence, and a buffer formulated to facilitate activity of the MSEs and the DNA polymerase.
Integrated MSE cleavage and quantitative PCR in same reaction volume
Integrated MSE cleavage followed by quantitative PCR amplification in the same reaction mixture without adjusting the reaction volume [procedural detail omitted for safety].
Quantitation of methylation by amplified product
Quantitating the proportion of methylation in the DNA sequence of the DNA sample by quantitating the amplified product.
Use of thermally stable DNA polymerase
Inclusion of a thermal stable DNA polymerase in the premixture to enable the claimed integration of endonuclease activity and quantitative PCR.
Claim 1 covers a one‑pot premixture defined by specified MSEs (AccII, HpaII, HpyCH4IV), a thermally stable polymerase, primers and buffer, and an integrated workflow that performs MSE cleavage and qPCR in the same reaction volume with methylation quantitation based on the amplified product.
Stated Advantages
Single reaction system integrating MSE specificity with real-time PCR for rapid and accurate DNA methylation quantification.
Decreased opportunity for contamination by combining multiple steps into one reaction.
Cost-effective and region-specific methylation status quantification suitable as a tool in research and diagnostics.
Documented Applications
Detecting or quantifying DNA methylation at a locus using methylation-sensitive endonucleases formulated with a polymerase in a reaction mixture.
Quantitating the proportion of DNA methylation in a DNA sequence or at a specific DNA position, including site-specific DNA methylation prevalence in a genomic DNA sample.
Provision of kits and reagents comprising reaction mixtures, control and test premixes, oligonucleotide primers, and reference DNA samples for performing the disclosed methylation methods.
Analysis of mammalian genomic DNA (including human genomic DNA) obtained from sources such as a human subject, a cell line, or a tissue bank and sample types including blood, tissue biopsy, urine, or saliva.
Use in research and diagnostics for rapid and accurate methylation profiling.
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