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Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation

Nature Protocols volume 2, pages 1931–1936 (2007)Cite this article

Abstract

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.

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Figure 1
Figure 2: Detailed outline of methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) assay showing specific amplification of bisulfite-converted DNA target sequences after SNuPE, electrophoresis and analysis of Ms-SNuPE reaction products.
Figure 3: Example of methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) assay.

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References

  1. Laird, P.W. & Jaenisch, R. The role of DNA methylation in cancer genetic and epigenetics. Annu. Rev. Genet. 30, 441–464 (1996).

    Article  CAS  Google Scholar 

  2. Gaudet, F. et al. Induction of tumors in mice by genomic hypomethylation. Science 300, 489–492 (2003).

    Article  CAS  Google Scholar 

  3. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    Article  CAS  Google Scholar 

  4. Jones, P.A. & Baylin, S.B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3, 415–428 (2002).

    Article  CAS  Google Scholar 

  5. Jones, P.A. & Baylin, S.B. The epigenomics of cancer. Cell 128, 683–692 (2007).

    Article  CAS  Google Scholar 

  6. Feinberg, A.P. & Vogelstein, B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301, 89–92 (1983).

    CAS  PubMed  Google Scholar 

  7. Singer-Sam, J., LeBon, J.M., Tanguay, R.L. & Riggs, A.D. A quantitative HpaII-PCR assay to measure methylation of DNA from a small number of cells. Nucleic Acids Res. 18, 687(1990).

    Article  CAS  Google Scholar 

  8. Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D. & Baylin, S.B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93, 9821–9826 (1996).

    Article  CAS  Google Scholar 

  9. Kuppuswamy, M.N. et al. Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes. Proc. Natl. Acad. Sci. USA 88, 1143–1147 (1991).

    Article  CAS  Google Scholar 

  10. Singer-Sam, J., LeBon, J.M., Dai, A. & Riggs, A.D. A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide. PCR Methods Appl. 1, 160–163 (1992).

    Article  CAS  Google Scholar 

  11. Szabo, P.E. & Mann, J.R. Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms. Genes Dev. 9, 3097–3108 (1995).

    Article  CAS  Google Scholar 

  12. Greenwood, A.D. & Burke, D.T. Single nucleotide primer extension: quantitative range, variability, and multiplex analysis. Genome Res. 6, 336–348 (1996).

    Article  CAS  Google Scholar 

  13. Gonzalgo, M.L. & Jones, P.A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25, 2529–2531 (1997).

    Article  CAS  Google Scholar 

  14. Saito, Y. et al. Specific activation of microRNA-127 with downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells. Cancer Cell 9, 435–443 (2006).

    Article  CAS  Google Scholar 

  15. Liang, G., Robertson, K.D., Talmadge, C., Sumegi, J. & Jones, P.A. The gene for a novel transmembrane protein containing epidermal growth factor and follistatin domains is frequently hypermethylated in human tumor cells. Cancer Res. 60, 4907–4912 (2000).

    CAS  PubMed  Google Scholar 

  16. Cheng, J.C. et al. Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J. Natl. Cancer Inst. 95, 399–409 (2003).

    Article  CAS  Google Scholar 

  17. Cheng, J.C. et al. Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol. Cell. Biol. 24, 1270–1278 (2004).

    Article  CAS  Google Scholar 

  18. Egger, G. et al. Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival. Proc. Natl. Acad. Sci. USA 103, 14080–14085 (2006).

    Article  CAS  Google Scholar 

  19. Liang, G. et al. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol. Cell. Biol. 22, 480–491 (2002).

    Article  CAS  Google Scholar 

  20. Kaminsky, Z.A., Assadzadeh, A., Flanagan, J. & Petronis, A. Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions. Nucleic Acids Res. 33, e95 (2005).

    Article  Google Scholar 

  21. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  Google Scholar 

  22. Fatemi, M. et al. Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level. Nucleic Acids Res. 33, e176 (2005).

    Article  Google Scholar 

  23. Markl, I.D. & Jones, P.A. Presence and location of TP53 mutation determines pattern of CDKN2A/ARF pathway inactivation in bladder cancer. Cancer Res. 58, 5348–5353 (1998).

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Urology, James Buchanan Brady Urological Institute, The Johns Hopkins Medical Institutions, 600 North Wolfe Street, Baltimore, 21287, Maryland, USA

    Mark L Gonzalgo

  2. Department of Urology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, NOR 7338, 1441 Eastlake Avenue, Los Angeles, 90089, California, USA

    Gangning Liang

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  1. Mark L Gonzalgo
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Correspondence to Mark L Gonzalgo or Gangning Liang.

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Competing interests

M.L.G. and G.L. have a patent (United States patent 6,811,982) entitled “A cancer diagnostic method based upon DNA methylation differences.“

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Gonzalgo, M., Liang, G. Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation. Nat Protoc 2, 1931–1936 (2007). https://doi.org/10.1038/nprot.2007.271

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