Services

The DNA Methylation Analysis Facility is funded by the Center for Cancer Epigenetics and the Department of Epigenetics and Molecular Carcinogenesis. It provides services related to the analysis of DNA methylation for researchers across all MD Anderson campuses and in the Texas Medical Center. Services include: consultation with investigators to choose the best method of analysis; sample preparation, assay design, and sample processing for the most commonly used gene-specific and genome-wide (PyroMeth, RRBS and Methyl-Seq) DNA methylation analyses; developing and maintaining a library of commonly used assays to measure global DNA methylation (LINE, Alu) and common gene-specific tests (p16, e-cadherin, MLH1, p15 and others). The core is located in the Mitchell Basic Science Research Building on the MD Anderson Cancer Center campus in Houston. We are fully equipped for molecular biology research and have a Qiagen Pyrosequencing machine.

The main obstacle to DNA methylation analysis is that methylated cytosines cannot be detected simply by sequencing. During PCR amplification, methylated cytosines are not differentiated by the DNA polymerase and like unmethylated cytosines, they are paired with guanosine dinucleotides. Thus, capturing methylated cytosines depends on indirect methods. The most commonly used are (1) restriction enzyme-based approaches, which take advantage of methylation-sensitive enzymes; (2) affinity-based approaches, in which either antibodies against 5-methylcytosine or against methyl-binding domain proteins are used to collect the methylated fraction of the genome, and (3) bisulfite conversion of non-methylated cytosines to thymidine through a hydrolytic deamination reaction, which takes advantage of the non-reactivity of methylated cytosines to free hydroxyl groups. Each one of these methods has an important application in studying the epigenome and has been individually or combinatorially applied to individual genes and large-scale analyses. Among these methods, bisulfite conversion is the gold-standard due to its high resolution when combined with sequencing methods. In this way, every single cytosine can be identified as methylated or unmethylated.

The choice of an appropriate DNA methylation analysis method depends on whether the investigation is still in the discovery stage or whether a set of targets has already been decided. Further, special considerations regarding assay design and detection methods must be addressed when dealing with minimal amounts of DNA or when applying DNA methylation as a molecular marker. The DNA Methylation Analysis Facility will work with investigators to design assays and conduct experiments. To arrange a consultation, please contact Marcos Estécio, Ph.D., at mestecio@mdanderson.org.

Researchers with projects that involve next-generation sequencing services should always consult a bioinformatician before planning the detailed project. For assistance with bioinformatics analyses, MD Anderson users may consult with their institutionally assigned, departmental bioinformatician, seek advice on potential bioinformatics collaborators from the Epicore directors, or contact Arif Harmanci, Ph.D., of UT Health by email.

Similar to pyrosequencing, in bisulfite sequencing, gene-specific targets are amplified in a PCR reaction with primers that produce amplicons from both methylated and unmethylated alleles. Sequencing of individually cloned products by Sanger sequencing can reveal the methylation status of dozens of sites and identify allelic-specific methylation.

For scheduling DNA Methylation Analysis services, please use iLab