Development of a DNA Methylation Assay Using an Engineered Methyl-CpG-Binding Domain Conference: International Conference on Epigenetics and BioengineeringYear: 2017Proceeding: International Conference on Epigenetics and BioengineeringGroup: General SubmissionsSession: Detecting epigenetic modifications (DNA, RNA, histones) Time: Thursday, December 14, 2017 - 12:50pm-1:15pm Authors: Tam, B. E., Massachusetts Institute of Technology Hao, Y., Massachusetts Institute of Technology Sung, K. J., Massachusetts Institute of Technology Dabbousi, D. B., Massachusetts Institute of Technology Sikes, H. D., Massachusetts Institute of Technology Improved DNA methylation detection methods are needed in order to expand access to methylation assays for cancer patients. Currently, clinical methylation assessment uses Methylation-Specific PCR (MS-PCR), which relies on bisulfite conversion of unmethylated cytosine bases. Bisulfite-based methods require careful optimization to prevent false positives or negatives,1 and it has also been shown that 12% of tests give inconclusive results,2 meaning that the results differ between two replicates from the same sample. Protein-based assays that use a binding protein to recognize methylated CpG dinucleotides not only eliminate the need for bisulfite conversion, but they also can provide additional information about the degree of methylation rather than the simple âyesâ or ânoâ result obtained from MS-PCR. These methods often use the Methyl-CpG-Binding Domain (MBD) family of proteins, most of which bind specifically to double-stranded DNA with symmetrically methylated CpG dinucleotides (the cytosine is methylated on both strands). We have used directed evolution to develop an MBD variant that binds to hemi-methylated DNA (where one of the two strands is methylated) but not unmethylated DNA. Evaluation of the binding affinity of the engineered protein to a DNA sequence with a single hemi-methylated CpG site showed a dissociation constant of 5.6 ± 1.4 nM.3 Using this engineered protein, we have developed a method to profile the methylation status of the MGMT promoter. In this assay, the sequence of interest is captured by hybridization to an unmethylated DNA probe and the methylation status of the target DNA is determined by MBD binding. This assay has significant advantages over those that require methylated DNA probes because unmethylated DNA probes are easy and inexpensive to produce, allowing probe optimization in assay development and adaptation of the method for other target sequences to be completed more quickly. 1 D. P. Genereux, W. C. Johnson, A. F. Burden, R. StÃ¶ger and C. D. Laird, Nucleic Acids Res., 2008, 36, e150. 2 D. Xia, D. A. Reardon, J. L. Bruce and N. I. Lindeman, J. Mol. Diagnostics, 2016, 18, 864â871. 3 B. E. Tam, K. Sung and H. D. Sikes, Mol. Syst. Des. Eng., 2016, 1, 273â277.