Abstract
We describe a highly reproducible and multiplexed method, the GoldenGate assay for methylation, for high-throughput quantitative measurements of DNA methylation. It can analyze up to 1,536 targeted CpG sites in 96 samples simultaneously, using only 250 ng of genomic DNA. The method is akin to a “genotyping” of bisulfite-converted DNA. Assay probes can be designed to interrogate the Watson strand, the Crick strand, or both strands at each CpG site. Assay end products are processed using Illumina universal bead arrays. As a result, gene or CpG sets can be refined iteratively–-no custom arrays need to be developed. This method allows the detection of as little as 2.5% methylation, and can distinguish 17% difference in absolute methylation level between samples. The method is highly reproducible and compares very well with other common methods of methylation detection, such as methylation-specific PCR and bisulfite sequencing. The Illumina GoldenGate Methylation technology should prove useful for DNA methylation analyses in large populations, with potential application to biomarker discovery and validation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Reference
Feinberg, A. P., Tycko, B. (2004) The history of cancer epigenetics. Nat Rev Cancer 4, 143–153.
Jones, P. A., Martienssen, R. (2005) A blueprint for a Human Epigenome Project: the AACR Human Epigenome Workshop. Cancer Res 65, 11241–11246.
Bibikova, M., Lin, Z., Zhou, L., et al. (2006) High-throughput DNA methylation profiling using universal bead arrays. Genome Res 16, 383–393.
Fan, J. B., Oliphant, A., Shen, R., et al. (2003) Highly parallel SNP genotyping. Cold Spring Harb Symp Quant Biol 68, 69–78.
Fan, J. B., Gunderson, K. L., Bibikova, M., et al. (2006) Illumina universal bead arrays. Methods Enzymol 410, 57–73.
Lin, Z., Thomas, N. J., Bibikova, M., et al. (2007) DNA methylation markers of surfactant proteins in lung cancer. Int J Oncol 31, 181–191.
Bjornsson, H. T., Brown, L. J., Fallin, M.D., et al. (2007) Epigenetic specificity of loss of imprinting of the IGF2 gene in Wilms tumors. J Natl Cancer Inst 99, 1270–1273.
Bibikova, M., Chudin, E., Wu, B., et al. (2006) Human embryonic stem cells have a unique epigenetic signature. Genome Res 16, 1075–1083.
Barker, D. L., Theriault, D., Che, D., et al. (2003) Self-assembled random arrays: high-performance imaging and genomics applications on a high-density microarray platform, in (Nicolau, D. V., Raghavachari, R., eds.), Microarrays and Combinatorial Technologies for Biomedical Applications: Design, Fabrication, and Analysis, Proceedings of SPIE, 4966, pp. 1–11.
Gunderson, K. L., Kruglyak, S., Graige, M. S., et al. (2004) Decoding randomly ordered DNA arrays. Genome Res 14, 870–877.
Wang, R. Y., Gehrke, C. W., Ehrlich, M. (1980) Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res 8, 4777–4790.
Grunau, C., Renault, E., Roizes, G. (2002) DNA Methylation Database “MethDB”: a user guide. J Nutr 132, 2435S– 2439S.
Rakyan, V. K., Hildmann, T., Novik, K. L., et al. (2004) DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol 2, e405.
Eckhardt, F., Lewin, J., Cortese, R., et al. (2006) DNA methylation profiling of human chromosomes 6, 20 and 22. Nat Genet 38, 1378–1385.
Herman, J. G., Graff, J. R., Myohanen, S., et al. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93, 9821–9826.
Adorjan, P., Distler, J., Lipscher, E., et al. (2002) Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Res 30, e21.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bibikova, M., Fan, JB. (2009). GoldenGate® Assay for DNA Methylation Profiling. In: Tost, J. (eds) DNA Methylation. Methods in Molecular Biology, vol 507. Humana Press. https://doi.org/10.1007/978-1-59745-522-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-522-0_12
Publisher Name: Humana Press
Print ISBN: 978-1-934115-61-9
Online ISBN: 978-1-59745-522-0
eBook Packages: Springer Protocols