Natalia Kholod, Jacques Boniver, Philippe Delvenne

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Presentation transcript:

A New Dimethyl Sulfoxide-Based Method for Gene Promoter Methylation Detection Natalia Kholod, Jacques Boniver, Philippe Delvenne The Journal of Molecular Diagnostics Volume 9, Issue 5, Pages 574-581 (November 2007) DOI: 10.2353/jmoldx.2007.070025 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Analysis of DAPK promoter methylation in human cervical keratinocytes. Isolated DNA was methylated in vitro by SssI methylase and compared with the same untreated DNA preparation. A: Ms-DMSO-PCR: 15 ng of each DNA template were taken for each PCR reaction with the concentration of DMSO ranging from 0 to 8%. B: PCR of MspI- and HpaII-digested DNA. N, noncut DNA; M, MspI digest; H, HpaII digest; L, 100-bp DNA ladder (Fermentas). The Journal of Molecular Diagnostics 2007 9, 574-581DOI: (10.2353/jmoldx.2007.070025) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Ms-DMSO-PCR of TIMP3, RASSF1A, and MGMT gene promoters. CaSki cell DNA was methylated in vitro by SssI methylase and compared with the same untreated DNA preparation. Ten ng of each DNA template were taken for each PCR reaction with the concentration of DMSO ranging from 1 to 7%. L, 100-bp DNA ladder. The Journal of Molecular Diagnostics 2007 9, 574-581DOI: (10.2353/jmoldx.2007.070025) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Sequence region of the DAPK promoter. All potentially methylated CG sites are labeled with an asterisk. The primer sequences used for the amplification of different parts of the promoter and the starting point of transcription are indicated. The Journal of Molecular Diagnostics 2007 9, 574-581DOI: (10.2353/jmoldx.2007.070025) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Methylation analysis of different fragments of DAPK promoter. CaSki cell DNA was methylated in vitro by SssI methylase and compared with the same untreated DNA preparation. Ms-DMSO-PCR: 5 to 10 ng of each DNA template were taken for each PCR reaction with the concentration of DMSO ranging from 1 to 5%. L, 100-bp DNA ladder. Primers and their location in promoter region are shown in Figure 3 and Table 2. The Journal of Molecular Diagnostics 2007 9, 574-581DOI: (10.2353/jmoldx.2007.070025) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Analysis of DAPK promoter methylation in the SiHa cell line. A: PCR of MspI- and HpaII-digested DNA of methylated in vitro by SssI methylase and the same untreated preparation. N, Noncut DNA; M, MspI digest; H, HpaII digest; L, 100-bp DNA ladder. B: Ms-DMSO-PCR of the same samples: 10 ng of each DNA template were taken for each PCR reaction with the concentration of DMSO ranging from 1 to 6%. C: PCR of MspI- and HpaII-digested DNA from 5-aza-dC or mock-treated SiHa cells. N, Noncut DNA; M, MspI digest; H, HpaII digest; L, 100-bp DNA ladder. D: Ms-DMSO-PCR of the same samples: 10 ng of each DNA template were taken for each PCR reaction with the concentration of DMSO ranging from 1 to 5%. The Journal of Molecular Diagnostics 2007 9, 574-581DOI: (10.2353/jmoldx.2007.070025) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Sensitivity of Ms-DMSO-PCR to time course methylation and detection of methylation in CaSki and SiHa DNA mixtures. A: Ms-DMSO-PCR of DAPK promoter of DNA from CaSki cells methylated in vitro for 0, 30, 60, and 120 minutes: 10 ng of each DNA template were taken for each PCR reaction with the concentration of DMSO ranging from 1 to 5%. B: PCR of MspI- and HpaII-digested DNA of the same samples. N, Noncut DNA; M, MspI digest; H, HpaII digest. C: Ms-DMSO-PCR of DAPK promoter from CaSki and SiHa DNA mixtures: 10 ng of each DNA template or mixture were taken for each PCR reaction with the concentration of DMSO ranging from 1 to 4%. The Journal of Molecular Diagnostics 2007 9, 574-581DOI: (10.2353/jmoldx.2007.070025) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 7 Ms-DMSO-PCR of DAPK and MGMT promoters in clinical samples of the uterine cervix: 10 ng of each DNA template were taken for each PCR reaction with the concentration of DMSO ranging from 2 to 5% for frozen biopsies and 0 to 2% for FFPE tissue. Nl cells, HPV−, normal cells, HPV-negative; SCC HPV+, squamous cell carcinoma, HPV-positive. A: Demethylation of DAPK promoter in frozen biopsies of SCC. B: Methylation of MGMT promoter in frozen biopsies of SCC. C: Demethylation of DAPK promoter in FFPE tissues of SCC. The Journal of Molecular Diagnostics 2007 9, 574-581DOI: (10.2353/jmoldx.2007.070025) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions