by Caitlin Smith
We all know that DNA mutations can cause phenotypic changes—but what about genetic regulations that aren’t caused by a sequence change, so-called epigenetic changes? One important type is DNA methylation. A potent regulator of gene expression, it’s the biochemical addition of methyl groups to cytosines in cytosine-phosphate-guanosine (CpG) dinucleotides. Such a simple mechanism can have dire consequences—many cancers show aberrant methylation patterns, and methylation is central to many other important processes, such as embryonic development and cell cycle regulation.
Randy Jirtle, a professor in the department of radiation oncology at Duke University, studies the role of genomic imprinting in human diseases by looking at inherited regulatory marks consisting of parent-specific DNA methylation. “We need to define the human epigenome as a function of tissue type and stage of life (such as pre- and post-natally, puberty, and old age) in order to understand how genetically identical twins often vary in disease susceptibility, and chimpanzees can be markedly different from humans even though our genomes are quite similar,” says Jirtle. This seems a tall order for the relatively simple addition of a methyl group to a base. So how do epigenetic researchers study DNA methylation today?
Though many techniques have been developed for detecting DNA methylation—methylation-sensitive restriction enzymes, methyl-binding proteins, and anti-methylcytosine antibodies, for example—only one technique, bisulfite conversion, has recently become a favored contender, and so will be the focus of this article. In this method, sodium bisulfite is used to convert unmethylated cytosines to uracils. Thus the methylated sites can be distinguished on the basis of sequence, and can be detected by various methods including methylation-specific PCR (MSP), or sequencing after PCR amplification.
Bisulfite conversion: the gold standard—with caveats
Indeed, one of the challenges facing scientists is that there is no one best method to use for detection of DNA methylation. For example, they use different methods if they are doing global profiling of methylation patterns, or studying individual loci or specific methylated cytosines. “The biggest challenge is coming up with a technology that has broad applications,” says Christopher Adams, research and development manager of epigenetics at Invitrogen. “Currently, multiple technologies are needed to perform this type of comprehensive analysis.” Though bisulfite conversion is seen by many as the best technique, only now are its limitations being overcome.
Adams explains that “the gold standard for methylation analysis, bisulfite conversion, has some challenges associated with it. This type of analysis damages the DNA, is labor intensive, and technically challenging. It's a difficult technology to work with, but currently the best thing out there.” Researchers at Zymo Research have strived to fine tune the bisulfite conversion method by improving the conversion efficiency and minimizing DNA degradation. Says Marc Van Eden, scientist and liaison at Zymo Research: “Essential to this process was the ability to achieve nearly 100% conversion of unmethylated cytosine into uracil while maximizing the recovery of input DNA. Our refinement of the bisulfite conversion process has simplified the procedure and made the technology more reliable.”
Scientists at Human Genetic Signatures claim to have advanced the bisulfite conversion method further. “ Our original MethylEasy™ product range revolutionized the bisulfite conversion market by introducing truly lossless technology whereby the original DNA was not degraded during the conversion process,” says John Melki, senior principal research scientist at Human Genetic Signatures. Their newest product, the MethylEasy™ Xceed bisulfite conversion kit, improves on this with a 90-minute run-time (compared to 6-18 hours for the original protocol), and an “exceptional sensitivity that requires only 50 pg of starting material (equivalent to only 8 mammalian cells),” says Melki.
Qiagen is attempting to address the integrated workflow issue with their improved EpiTect product line. “From May 2008 on, Qiagen will offer the first complete and standardized workflow for reliable methylation analysis ... from DNA sample collection, stabilization and purification, to bisulfite conversion and real-time or endpoint PCR methylation analysis or sequencing,” says Gerald Schock, global product manager of whole genome amplification and epigenetics at Qiagen. Qiagen’s EpiTect DNA Protect technology ensures complete bisulfite conversion, and the new buffers in their EpiTect MethyLight PCR Kits increase the stability of bound primers and probes. “For increased reliability in end-point MSP, Qiagen has developed the EpiTect MSP Kit, which includes a genetically engineered HotStarTaq d-Tect polymerase for increased MSP primer extension specificity,” says Schock. “The EpiTect Whole Bisulfitome Kit includes proven Multiple Displacement Amplification (MDA) based REPLI-g technology which has been adapted to the special requirements of bisulfite-converted DNA for reliable amplification of the entire bisulfite-converted genome—the bisulfitome.”
HRM: a new and sensitive method
A new approach to methylation detection after bisulfite conversion is high resolution melt (HRM) analysis. According to Brant Bassam, marketing manager for Corbett Life Science, HRM’s simplicity and high reproducibility make it an ideal method for methylation analysis. “HRM is a close-tube, post-PCR method that monitors the comparative disassociation (melting) behavior of different sequences,” says Bassam. “Following bisulfite treatment, methylated and unmethylated DNA samples contain different sequences, making them readily amenable to HRM analysis.” The technique uses Corbett’s Rotor-Gene 6000, “the ideal instrument for low-level methylation detection,” says Bassam, “as its unique rotary design provides the highest thermal and optical precision of any commercial system.” Bassam believes that the biggest challenge is producing consistent, fast and cost-effective methylation results, but that HRM can deliver this: “HRM methylation is an extremely methylation-sensitive, reliable, and cost-effective method. It can discriminate the proportional status of locus-specific methylation versus non-methylation and readily distinguish heterogeneous methylation status.”
Genome-wide DNA methylation analysis
With the advent of high-throughput sequencing-based technologies, many groups are turning their attention to developing genome-wide DNA methylation detection technologies, including Zymo Research. “We believe that determining genome-wide DNA methylation status will be essential when addressing how DNA methylation participates in aging, development, oncogenesis, and pathogenesis,” says Van Eden. One example of this is a soon-to-be-launched enrichment kit for methylated DNA based on monoclonal antibody enrichment from Zymo Research. This type of kit is meant to facilitate the development of high-throughput platforms or arrays for genome-wide methylation detection. “The monoclonal antibodies we have developed against 5-methylcytosine exhibit the highest levels of specificity that we have tested to date,” says Van Eden.
Epigenetic information as biomarkers for disease
Indeed, a global view of genetic regulation is increasingly important, according to Jirtle: “It is no longer wise to focus our research efforts primarily on genetic mutations, when it is becoming clear that deregulation of the epigenome early in development plays a major role in adult disease susceptibility.” Perhaps the most stirring results of the advances in methylation detection are the ever-closer clinical realities. “The most exciting application is the discovery of methylation biomarkers for numerous diseases including cancer,” says Adams. Schock agrees: “Developing biomarkers as diagnostic tools which enable detection of early stages of diseases such as cancer might be the most exciting area for the near future.”