CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics Antonia R. Sepulveda, Dan Jones, Shuji.

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CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics Antonia R. Sepulveda, Dan Jones, Shuji Ogino, Wade Samowitz, Margaret L. Gulley, Robin Edwards, Victor Levenson, Victoria M. Pratt, Bin Yang, Khedoudja Nafa, Liying Yan, Patrick Vitazka The Journal of Molecular Diagnostics Volume 11, Issue 4, Pages 266-278 (July 2009) DOI: 10.2353/jmoldx.2009.080125 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Bisulfite modification of DNA for methylation assays. Bisulfite modification converts unmethylated cytosine to uracil, while methylated cytosine is not modified. After PCR, uracil is replaced by thymine on the newly synthesized DNA strands. The Journal of Molecular Diagnostics 2009 11, 266-278DOI: (10.2353/jmoldx.2009.080125) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Methylation of the MLH1 gene CpG island promoter region detected by methylation specific PCR (MSP). After sodium bisulfite conversion of genomic DNA from colon cancer tumor samples (T1 and T2) or positive control DNA (Pos. Cont.), PCR was performed with the primer pair specific for methylated MLH1 DNA (M) or with the primer pair specific for the unmethylated MLH1 sequence (U). The negative control is a PCR reaction without DNA template. The DNA size ladder (Bp) is indicated. The presence of a PCR product in the lanes labeled (M) indicates the presence of CpG methylation in the sample T1 and in the positive control. For sample T2 no MLH1 CpG methylation is detected. The Journal of Molecular Diagnostics 2009 11, 266-278DOI: (10.2353/jmoldx.2009.080125) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Methylation-sensitive restriction enzymes for detection of CpG Methylation without bisulfite conversion. DNA is first treated with HhaI methylation-sensitive restriction endonuclease and then used for PCR. When the CpG locus being amplified is not methylated, HhaI cleaves its restriction site, resulting in lack of PCR amplification; whereas, if it is methylated, the HhaI restriction sites are protected from restriction enzyme digestion, allowing for PCR amplification. The Journal of Molecular Diagnostics 2009 11, 266-278DOI: (10.2353/jmoldx.2009.080125) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions