Amplicon region as epigenetic marker for the identification of non-classical monocytes
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11680294-B2
Publication Date
2023-06-20
Expiration Date
Abstract
The present invention relates to a method, in particular an in vitro method, for identifying non-classical monocytes, comprising analyzing the methylation status of at least one CpG position in the mammalian genomic region comprising an amplicon, wherein a demethylation or lack of methylation of said region is indicative for a non-classical monocyte, when compared to a classical monocyte or a non-monocyte cell. The analyses according to the invention can identify non-classical monocytes on an epigenetic level and distinguish them from all other cells in complex samples, such as, for example, other blood or immune cells. The present invention furthermore provides an improved method for quantifying non-classical monocytes, in particular in complex samples. The method can be performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue.
Core Innovation
The invention relates to producing and detecting an amplicon from bisulfite treated DNA using a mammalian genomic region of SEQ ID NO: 1 prior to bisulfite treatment. Genomic DNA from a mammalian cell sample is bisulfite treated to generate bisulfite treated DNA, and an amplicon is produced by amplifying a region comprising the mammalian genomic region of SEQ ID NO: 1 prior to bisulfite treatment. Amplifying is performed with polymerase chain reaction (PCR), and the amplicon is detected using a probe comprising the nucleic acid sequence of SEQ ID NO: 8.
The method characterizes the detected amplicon by requiring cytosine features at CA positions relative to SEQ ID NO: 2. The amplicon comprises CA at CA positions 242 and 248 relative to SEQ ID NO: 2 and one or more CA at a CA position selected from positions 39, 89, 123, 169, and 206 relative to SEQ ID NO: 2.
The method is positioned for epigenetic identification and quantification in non-classical monocytes by analyzing bisulfite accessibility through a TpG- and CpG-specific qPCR format. It distinguishes non-classical monocytes from classical monocytes and non-monocyte cells in complex samples by using probe systems and CpG position subsets within the AMP2213 genomic amplicon region. The disclosed approach is implemented as a kit and can include ratio-based quantification using an internal control gene such as GAPDH.
Claims Coverage
The provided content covers one independent claim and dependent refinements. The coverage centers on bisulfite treating mammalian genomic DNA, PCR-amplifying a defined region corresponding to SEQ ID NO: 1, probe-based detection using SEQ ID NO: 8, and CA-position requirements relative to SEQ ID NO: 2, with additional features including qPCR format and omission of purification and/or enrichment.
Bisulfite treating genomic DNA and PCR amplifying SEQ ID NO: 1 region prior to bisulfite treatment
A method comprising bisulfite treating isolated genomic DNA from a mammalian cell sample to generate bisulfite treated DNA, and producing an amplicon by amplifying from the bisulfite treated DNA a region comprising the mammalian genomic region of SEQ ID NO: 1 prior to bisulfite treatment, wherein the amplifying is performed with a polymerase chain reaction (PCR).
Probe-based detection using SEQ ID NO: 8
Detecting the amplicon with a probe comprising the nucleic acid sequence of SEQ ID NO: 8.
CA position constraints at CA positions 242 and 248 and selected additional CA positions
The amplicon comprises CA at CA positions 242 and 248 relative to SEQ ID NO: 2 and one or more CA at a CA position selected from positions 39, 89, 123, 169, and 206 relative to SEQ ID NO: 2.
qPCR amplification
The PCR performed is quantitative PCR (qPCR).
Omission of purifying and/or enriching the cell sample
The method is performed without a step of purifying and/or enriching the cell sample.
The claim coverage is anchored in bisulfite treatment of mammalian genomic DNA followed by PCR amplification of a region corresponding to SEQ ID NO: 1 and detection with a SEQ ID NO: 8 probe, with CA-position constraints relative to SEQ ID NO: 2. Dependent features further narrow the approach to qPCR and allow performance without purifying and/or enriching the cell sample.
Stated Advantages
Allows detection of methylation-associated cytosines from bisulfite treated DNA using probe-based detection of an amplicon.
Enables discrimination of non-classical monocytes from classical monocytes and non-monocyte cells in complex samples.
Supports epigenetic identification and quantification using TpG- and CpG-specific qPCR formats and selected CpG position subsets.
Supports ratio-based quantification using an internal control gene such as GAPDH.
Provided in a kit format.
Documented Applications
Epigenetic identification/quantification of non-classical monocytes, including distinguishing non-classical monocytes from classical monocytes and non-monocyte cells in complex samples such as whole blood or non-trypsinized tissue.
Interested in licensing this patent?