Assays to determine DNA methylation and DNA methylation markers of cancer
Inventors
Guo, Wei • PIATTI, Paolo • Yang, Xiaojing • JIA, Xi-Yu
Assignees
Publication Number
US-10801060-B2
Publication Date
2020-10-13
Expiration Date
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Abstract
Methods are provided for determining a genomic methylation profile in a DNA sample. In certain aspects, the methods can be used to determine if a subject has, or is at risk for developing, a bladder cancer or other cancers of the urinary tract. Methods for treatment of such subjects are likewise provided.
Core Innovation
Methods are provided for determining a genomic methylation profile in a DNA sample. In certain aspects, the methods can be used to determine if a subject has, or is at risk for developing, a bladder cancer or other cancers of the urinary tract, and methods for treatment of such subjects are likewise provided.
In a first embodiment, the invention provides a method comprising obtaining a substantially purified test genomic DNA sample; contacting a portion of the test genomic DNA with a reaction mixture comprising at least two methylation sensitive restriction endonucleases, a hot-start DNA polymerase, a pH buffered salt solution, dNTPs, DNA primer pairs for PCR amplification of at least first, second and third different genomic regions, and fluorescent probes complementary to sequences in said first, second and third different genomic regions, wherein each probe comprises a distinct fluorescent label; subjecting the reaction mixture to digestion and thermal cycling while detecting fluorescent signals, thereby performing real time PCR; and using the detected fluorescent signals to determine the genomic DNA methylation profile in a sample.
The invention further provides related reaction mixtures and kits, multiplex designs (including multiple test regions and internal controls) and alternative embodiments including methods using bisulfite-converted DNA and a DNA methylation standard curve. The disclosure describes marker panels (see Table 1A) and examples of primer and probe sets (Tables 1B and 1C), and notes that the reaction design allows multiplex digestion and qPCR in the same buffer and permits analysis of CpG methylation without treating the DNA with sodium bisulfite (procedural detail omitted for safety).
Claims Coverage
One independent claim is presented covering a reaction mixture for multiplex methylation analysis; five main inventive features are extracted.
reaction mixture composition
A reaction mixture comprising: (i) at least two methylation sensitive restriction endonucleases; (ii) a hot-start DNA polymerase; (iii) a pH buffered salt solution; (iv) dNTPs; and (v) a substantially purified genomic DNA sample.
multiplex amplification with fluorescent probes
DNA primer pairs for PCR amplification of at least a first, second and third different genomic region and fluorescent probes complementary to sequences in said first, second and third different genomic regions for quantitative detection of amplified sequences, wherein each of the probes comprises a distinct fluorescent label.
cleavage control region
A first genomic region that is a cleavage control known to be unmethylated.
copy number control region
A second genomic region that is a copy number control that does not include any cut site of the methylation sensitive restriction endonucleases of the reaction mixture.
test region with multiple cut sites
A third genomic region that is a test region having an unknown amount of methylation and including at least three cut sites of the methylation sensitive restriction endonucleases of the reaction mixture.
The independent claim covers a multiplex reaction mixture combining multiple methylation-sensitive restriction endonucleases, hot-start polymerase, distinct fluorescent probes, and a three-region design (cleavage control, copy number control, and a multi-cut-site test region) for quantitative determination of genomic DNA methylation profiles.
Stated Advantages
Allows for highly quantitative and rapid methods for determining the methylation profile from patient samples.
Methylation profiles can be used to determine disease risk, to determine if a patient has a cancer, and to assess the aggressiveness of a cancer.
Profiles can be used to select the most effective therapy for a subject having a disease.
Allows for the analysis of CpG methylation without treating the DNA with sodium bisulfite (procedural detail omitted for safety).
Described embodiments are characterized as robust, accurate, sensitive, non-invasive and very simple, with a very low detection limit (examples report sensitivity of 100% and specificity of 85.71% in a preclinical cohort).
Documented Applications
Determining if a subject has, or is at risk for developing, bladder cancer or other cancers of the urinary tract by determining a methylation status in one or more genomic regions (Table 1A).
Detecting the presence of, or an increased risk of, bladder cancer or other cancers of the urinary tract in a patient by determining methylation status in genomic regions and comparing to a reference level.
Monitoring bladder cancer recurrence or a risk of bladder cancer recurrence, including use in patients previously treated for or diagnosed with bladder cancer.
Treating a patient based on a determined genomic methylation profile, including performing a biopsy or administering an anti-cancer therapy such as chemotherapy, radiotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, cytokine therapy, or BCG therapy.
Use with DNA isolated from urine, stool, saliva, blood or tissue samples, including biopsy samples and urine samples, as explicitly described sample sources.
Providing kits for analysis of DNA methylation comprising primers or probes designed to detect methylation in genomic regions of Table 1A and reagents such as methylation sensitive restriction endonucleases and other assay components.
Determining copy number of methylated DNA molecules and potentially detecting chromosomal aneuploidy relative to one or more gene regions.
Use of marker panels (e.g., markers listed in Table 1A) and primer/probe sets (Tables 1B and 1C) for multiplex assays (CARE assay, MMSP assay) to detect cancer-associated methylation signatures in clinical samples.
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