Improved Quantification of DNA Methylation Using Methylation-Sensitive Restriction Enzymes and Real-Time PCR

Heterogeneity of cells with respect to the DNA methylation status at a specific CpG site is a problem when assessing methylation status. We have developed a simple two-step method for the quantification of the percent of cells that display methylation at a specific CpG site in the promoter of a specific gene. The first step is overnight digestion of genomic DNA (optimal conc. 20ng/5μl) with a relevant methylation-sensitive restriction enzyme (optimal 2 units). This is followed by real time PCR, using the SYBR® Green method, with primers that bracket the site cleaved by the enzyme. By including fully methylated and fully non-methylated DNA in each PCR plate, the errors caused by non-specific digestion or incomplete digestion can be measured and used to adjust the raw results and thus increase specificity. The method can detect differences in methylation status if these are more than 10%. No specialized equipment is required beyond the real-time PCR system and the method can be adapted for any of the 53 commercially available methylation-sensitive restriction enzymes.

[1]  H. Roach,et al.  PCR-based methods to determine DNA methylation status at specific CpG sites using methylation-sensitive restriction enzymes , 2007 .

[2]  T. Aigner,et al.  DNA methylation in osteoarthritic chondrocytes: a new molecular target. , 2007, Osteoarthritis and cartilage.

[3]  A. DeMarzo,et al.  Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation , 2006, Nucleic acids research.

[4]  H. Roach,et al.  Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions. , 2005, Arthritis and rheumatism.

[5]  B. Trock,et al.  Preoperative Serum DNA GSTP1 CpG Island Hypermethylation and the Risk of Early Prostate-Specific Antigen Recurrence Following Radical Prostatectomy , 2005, Clinical Cancer Research.

[6]  C. Denning,et al.  Stem-cell consequences of embryo epigenetic defects , 2004, The Lancet.

[7]  W. Dean,et al.  Epigenetic reprogramming during early development in mammals. , 2004, Reproduction.

[8]  A. Olek,et al.  A real-time PCR assay for DNA-methylation using methylation-specific blockers. , 2004, Nucleic acids research.

[9]  M. Szyf,et al.  Targeting DNA methylation in cancer , 2003, Ageing Research Reviews.

[10]  M. Ehrlich,et al.  Expression of various genes is controlled by DNA methylation during mammalian development , 2003, Journal of cellular biochemistry.

[11]  Y. Yuasa.  DNA methylation in cancer and ageing , 2002, Mechanisms of Ageing and Development.

[12]  Long-Cheng Li,et al.  MethPrimer: designing primers for methylation PCRs , 2002, Bioinform..

[13]  Christoph Grunau,et al.  Identification and resolution of artifacts in bisulfite sequencing. , 2002, Methods.

[14]  J. Herman,et al.  Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. , 2001, Human molecular genetics.

[15]  I. Pogribny,et al.  Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis. , 2000, Cancer research.

[16]  P. Laird,et al.  CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression. , 1999, Cancer research.

[17]  W. Isaacs,et al.  CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: a new prostate cancer biomarker. , 1997, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[18]  P. Laird,et al.  COBRA: a sensitive and quantitative DNA methylation assay. , 1997, Nucleic acids research.

[19]  J. Herman,et al.  Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  L. E. McDonald,et al.  A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Meehan,et al.  Methylation-sensitive polymerase chain reaction. , 2006, Methods in molecular biology.

[22]  T. Grange,et al.  MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome. , 2004, Nucleic acids research.

[23]  M. Monk.  Epigenetic programming of differential gene expression in development and evolution. , 1995, Developmental genetics.