Method for detecting methylation status by using methylation-independent primers
Inventors
Wojdacz, Tomasz Kazimierz • Hansen, Lise Lotte • Dobrovic, Alexander
Assignees
PETER MCCALLUM CANCER CENTRE • Aarhus University • Brainreader ApS • Peter Maccallum Cancer Centre
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Publication Number
US-8911937-B2
Publication Date
2014-12-16
Expiration Date
Abstract
A reliable and highly sensitive method is provided for detecting methylation status of CpG-containing nucleic acids by nucleic acid amplification and melting curve analysis of amplification products. The methods and compositions employs a novel design of primers, CpG-containing methylation-independent oligonucleotide primers, wherein both unmethylated and methylated alleles of a CpG-containing nucleic acid can be detected by use of only one set of primers after the CpG-containing nucleic acid has been subjected to cytosine to thymine conversion of unmethylated Cytosine. The method is useful for detection of methylation status in for example cancer genes and other disease related genes, wherein methylation influences gene expression.
Core Innovation
The invention relates to detecting the methylation status of a CpG-containing nucleic acid in a sample. The method includes denaturing the CpG-containing nucleic acid and treating it with bisulphite, which modifies at least one unmethylated cytosine in the CpG-containing nucleic acid, thereby producing a treated CpG-containing nucleic acid.
The method amplifies the treated CpG-containing nucleic acid using at least one methylation-independent oligonucleotide primer. The primer comprises within the first 10 nucleotides of the 5′-end at least one CpG dinucleotide in a region which hybridizes to the CpG-containing nucleic acid, and the primer hybridizes to both methylated and unmethylated CpG-containing nucleic acids after modification.
Methylation status is determined by melting curve analysis of the amplified CpG-containing nucleic acid, including high resolution melting. Primer design with CpG-containing behavior and annealing-temperature modulation is used to reduce PCR bias and enable sensitive, quantitative estimation of the methylated fraction from melting curves, including normalization of melting curves using a line-of-best-fit approach.
The disclosure further addresses heterogeneous methylation by introducing a digital MS-HRM concept for resolving allele-level heterogeneity via limiting dilution prior to melting-curve analysis. The described workflow is applied to methylation screening of multiple cancer-related and imprinting disorder genes, using sample types that include tissues and body fluids, and is supported by kit formats with control/reference mixtures of fully methylated and unmethylated materials.
Claims Coverage
Independent claim clm-00001 defines a complete workflow: denaturing CpG-containing nucleic acid, bisulphite treatment of unmethylated cytosines, amplification with a methylation-independent CpG-containing primer that hybridizes after modification to both methylated and unmethylated templates, and methylation calling by melting curve analysis. The provided claim set indicates a single independent claim (clm-00001), with dependent claims refining primer constraints, melting-curve analysis details, and quantitative/threshold relationships for methylation fraction.
CpG bisulphite modification and methylation-independent amplification
Amplifying the treated CpG-containing nucleic acid by means of at least one methylation-independent oligonucleotide primer, which comprises within the first 10 nucleotides of the 5′-end at least one CpG dinucleotide in a region which hybridizes to the CpG-containing nucleic acid, wherein the at least one methylation-independent oligonucleotide primer hybridizes to both methylated and unmethylated CpG-containing nucleic acids after modification.
Methylation determination by melting curve analysis
Analyzing the amplified CpG-containing nucleic acid by melting curve analysis.
Across the provided independent claim coverage, the key inventive combination is bisulphite modification of unmethylated cytosines followed by amplification using methylation-independent CpG-containing primers configured to hybridize to both methylated and unmethylated templates after modification, with methylation status determined by melting curve analysis of the amplified product.
Stated Advantages
Enables sensitive/quantitative estimation of methylated fraction based on melting-curve analysis.
Reduces PCR bias by configuring the CpG-containing primer behavior and annealing-temperature modulation.
Improves amplification across methylated and unmethylated alleles.
Supports detection of low methylation levels and quantitative methylation calling using normalized HRM curves.
Addresses heterogeneous methylation/allele-level heterogeneity using a digital MS-HRM concept.
Documented Applications
Methylation screening of multiple cancer-related and imprinting disorder genes, including MGMT and BNIP3.
Use with sample types including tissues and body fluids.
Kit formats including primers and control/reference mixtures (fully methylated/unmethylated) for standards and comparison.
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