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Fluorescent conjugated polymer-based FRET technique for detection of DNA methylation of cancer cells

Nature Protocols volume 5, pages 1255–1264 (2010)Cite this article

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Abstract

This protocol describes a homogeneous, convenient and sensitive DNA methylation detection method, using an optically amplifying cationic conjugated polymer (CCP, poly((1,4-phenylene)-2,7-[9,9-bis(6′-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide)). Genomic DNA from cancer cells is pretreated with a methylation-sensitive restriction endonuclease, followed by PCR amplification in the presence of fluorescein-labeled dNTP and Taq polymerase. The PCR only occurs for methylated DNA. DNA methylation of the gene sequence of interest is detected as a result of the fluorescence resonance energy transfer (FRET) between CCP and fluorescein that is incorporated into DNA. The methylated statuses of the p16, HPP1 and GALR2 promoters of five cancer cell lines (HT29, HepG2, A498, HL60 and M17) were assayed to provide an association study between the cancers and susceptibility genes, which shows great potential for early cancer diagnosis. This protocol simplifies previously available procedures by avoiding the need for primer labeling, isolation or purification steps, and sophisticated instruments. The assay takes about 20 h to obtain the methylated statuses of cancer cells.

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Figure 1: Schematic representation of CCP-based DNA methylation detection.
Figure 2
Figure 3: Analysis of PCR products for four genes (ActB, p16, HPP1 and GALR2) of five cancer cell lines (HT29, HepG2, A498, HL60 and M17) in 2% (wt/vol) agarose gel.
Figure 4: Fluorescence spectra measured in 1 ml of HEPES buffer (25 mM, pH 8.0) containing 0.2 μM CCP and 0.3% sample for analysis of genes (a) ActB, (b) p16, (c) HPP1 and (d) GALR2 in cell line HL60.
Figure 5: FRET ratios (I530nm/I424nm) for analysis of genes (a) ActB, (b) p16, (c) HPP1 and (d) GALR2 in five cell lines (HT29, HepG2, A498, HL60 and M17).
Figure 6: CCP-based quantitative analysis of DNA methylation.

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Acknowledgements

We are grateful to the National Natural Science Foundation of China (Nos. 90913014 and 20725308) and to the Major Research Plan of China (No. 2006CB932102).

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Authors and Affiliations

  1. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China

    Fude Feng, Libing Liu & Shu Wang

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  1. Fude Feng
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Contributions

F.F. conducted experiments and data analysis, and wrote the paper; L.L. conducted experiments and data analysis; S.W. designed experiments, performed data analysis and wrote the paper.

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Correspondence to Shu Wang.

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Feng, F., Liu, L. & Wang, S. Fluorescent conjugated polymer-based FRET technique for detection of DNA methylation of cancer cells. Nat Protoc 5, 1255–1264 (2010). https://doi.org/10.1038/nprot.2010.79

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