Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Sensitive digital quantification of DNA methylation in clinical samples

Abstract

Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers1,2. We have developed methyl-BEAMing technology to enable absolute quantification of the number of methylated molecules in a sample. Individual DNA fragments are amplified and analyzed either by flow cytometry3 or next-generation sequencing. We demonstrate enumeration of as few as one methylated molecule in 5,000 unmethylated molecules in DNA from plasma or fecal samples. Using methylated vimentin as a biomarker in plasma samples, methyl-BEAMing detected 59% of cancer cases. In early-stage colorectal cancers, this sensitivity was four times more than that obtained by assaying serum-carcinoembryonic antigen (CEA). With stool samples, methyl-BEAMing detected 41% of cancers and 45% of advanced adenomas. In addition to diagnostic and prognostic applications, this digital quantification of rare methylation events should be applicable to preclinical assessment of new epigenetic biomarkers and quantitative analyses in epigenetic research.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Digital quantification of rare DNA methylation in clinical samples by methyl-BEAMing.
Figure 2: Methyl-BEAMing to detect methylation of the vimentin gene using plasma from colorectal cancer patients.
Figure 3: Methyl-BEAMing to detect methylation of the vimentin gene using fecal DNA from colorectal tumor patients.

References

  1. 1

    Sidransky, D. Emerging molecular markers of cancer. Nat. Rev. Cancer 2, 210–219 (2002).

    CAS  Article  Google Scholar 

  2. 2

    Fleischhacker, M. & Schmidt, B. Circulating nucleic acids (CNAs) and cancer–a survey. Biochim. Biophys. Acta 1775, 181–232 (2007).

    CAS  PubMed  Google Scholar 

  3. 3

    Dressman, D., Yan, H., Traverso, G., Kinzler, K.W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. USA 100, 8817–8822 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Feinberg, A.P. & Tycko, B. The history of cancer epigenetics. Nat. Rev. Cancer 4, 143–153 (2004).

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

    Laird, P.W. The power and the promise of DNA methylation markers. Nat. Rev. Cancer 3, 253–266 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Diehl, F. et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc. Natl. Acad. Sci. USA 102, 16368–16373 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Diehl, F. et al. Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients. Gastroenterology 135, 489–498 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Bailey, V.J. et al. MS-qFRET: a quantum dot-based method for analysis of DNA methylation. Genome Res. 19, 1455–1461 (2009).

    CAS  Article  Google Scholar 

  10. 10

    Hayatsu, H. The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research. Mutat. Res. 659, 77–82 (2008).

    CAS  Article  Google Scholar 

  11. 11

    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).

    CAS  Article  Google Scholar 

  12. 12

    Xiong, Z. & Laird, P.W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25, 2532–2534 (1997).

    CAS  Article  Google Scholar 

  13. 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).

    CAS  Article  Google Scholar 

  14. 14

    Lo, Y.M. et al. Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction. Cancer Res. 59, 3899–3903 (1999).

    CAS  PubMed  Google Scholar 

  15. 15

    Eads, C.A. et al. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 28, E32 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Vogelstein, B. & Kinzler, K.W. Digital PCR. Proc. Natl. Acad. Sci. USA 96, 9236–9241 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Grunau, C., Clark, S.J. & Rosenthal, A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, E65 (2001).

    CAS  Article  Google Scholar 

  18. 18

    Okamoto, A. Chemical approach toward efficient DNA methylation analysis. Org. Biomol. Chem. 7, 21–26 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Zou, H. et al. Highly methylated genes in colorectal neoplasia: implications for screening. Cancer Epidemiol. Biomarkers Prev. 16, 2686–2696 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Chen, W.D. et al. Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene. J. Natl. Cancer Inst. 97, 1124–1132 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Jahr, S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61, 1659–1665 (2001).

    CAS  PubMed  Google Scholar 

  22. 22

    Diehl, F. et al. Circulating mutant DNA to assess tumor dynamics. Nat. Med. 14, 985–990 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Hundt, S., Haug, U. & Brenner, H. Comparative evaluation of immunochemical fecal occult blood tests for colorectal adenoma detection. Ann. Intern. Med. 150, 162–169 (2009).

    Article  Google Scholar 

  24. 24

    Lieberman, D.A. Screening for colorectal cancer. Clin. Cornerstone 4, 1–10 (2002).

    Article  Google Scholar 

  25. 25

    Kahi, C.J., Rex, D.K. & Imperiale, T.F. Screening, surveillance, and primary prevention for colorectal cancer: a review of the recent literature. Gastroenterology 135, 380–399 (2008).

    Article  Google Scholar 

  26. 26

    Humphrey, L.L., Helfand, M., Chan, B.K. & Woolf, S.H. Breast cancer screening: a summary of the evidence for the US Preventive Services Task Force. Ann. Intern. Med. 137, 347–360 (2002).

    Article  Google Scholar 

  27. 27

    Kent Moore, J., Smith, J.A., Whitney, D.H., Durkee, K.H. & Shuber, A.P. An electrophoretic capture method for efficient recovery of rare sequences from heterogeneous DNA. Biotechniques 44, 363–374 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Rago, C. et al. Serial assessment of human tumor burdens in mice by the analysis of circulating DNA. Cancer Res. 67, 9364–9370 (2007).

    CAS  Article  Google Scholar 

  29. 29

    Warnecke, P.M. et al. Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res. 25, 4422–4426 (1997).

    CAS  Article  Google Scholar 

  30. 30

    Diehl, F. et al. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat. Methods 3, 551–559 (2006).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank B. Berger for helpful discussions; D. Edelstein for the help with plasma collection; and M. Whalen and L. Kasturi for expert technical assistance. This work was supported by the Virginia and D.K. Ludwig Fund for Cancer Research; the Miracle Foundation; the Edelstein Fund; the US National Colorectal Cancer Research Alliance; The US National Institutes of Health grants CA43460,CA62924 and CA120237; The Danish Cancer Society; The Danish Research Council; and The Institute of Experimental Clinical Research, Aarhus University.

Author information

Affiliations

Authors

Contributions

M.L., S.D.M., S.N.G., K.W.K. and B.V. designed the project. M.L. developed the methyl-BEAMing assay and performed the experiments on plasma and fecal DNA. N.P., M.L. and Y.H. performed the Solexa sequencing experiments. H.M. performed the MSP assay. K.D. purified fecal DNA. S.N.G. provided statistical analysis. H.J., L.A.D., N.C.B., S.L., N.A. and K.S. collected clinical samples. W.-d.C., S.Z, V.E.V., F.D. and B.L. made intellectual contributions to the project. B.V., M.L. and S.D.M. wrote the manuscript.

Corresponding author

Correspondence to Sanford D Markowitz.

Ethics declarations

Competing interests

Technology for digital quantification of methylated DNA is the subject of a patent application from Johns Hopkins and Case Western Universities that include S.D.M., M.L., W.D.C., B.V. and K.W.K. as inventors. Under agreements between the Johns Hopkins University, Genzyme, Exact Sciences, Beckman, Inostics, and Invitrogen, K.W.K., B.V., M.L., and F.D. are entitled to a share of the royalties received by the University on sales of products related to BEAMing. Under agreements between Case Western University and Exact Sciences, S.D.M. and W.D.C. are entitled to a share of the royalties received on sales of products related to methylated vimentin DNA. Johns Hopkins University, K.W.K. and B.V. own stock in Genzyme and K.W.K., B.V., N.P., F.D. and L.D. own stock in Inostics, both of which are subject to certain restrictions under Johns Hopkins University policy. The terms of these arrangements are being managed by the universities in accordance with their conflict of interest policies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–5 (PDF 846 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, M., Chen, Wd., Papadopoulos, N. et al. Sensitive digital quantification of DNA methylation in clinical samples. Nat Biotechnol 27, 858–863 (2009). https://doi.org/10.1038/nbt.1559

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing