EpiGenie recently reviewed the text Epigenetics by Jorg Tost. Here is a more in depth summary of one the chapters provided courtesy of JA Gill who is a molecular biologist at NOAA in the Northwest Fisheries Science Center, to give you a little sneak peek into what the whole book is all about:
Methods for the Genome-wide and Gene-specific Analysis of DNA Methylation Levels and Patterns: by Jörg Tost
The editor of the book “Epigenetics” writes a chapter summarizing an assortment of new and traditional tools for the geneticist’s and molecular biologist’s toolbox for investigating DNA methylation. According to Tost, “No single method has emerged as the ‘gold’ standard unifying quantitative accuracy and high sensitivity or possibilities for whole-genome analysis and precise investigation of individual CpG positions.”
Tost’s statement reflects the revolutionary nature, uncertain and exciting, of these new technologies neatly summarized in this section. The focus is on DNA methylation. As a primer, the reader is briefed on some of the more traditional methods for detecting DNA methylation, such as mass spectrometric separation and chemical fission of radioactive labeled DNA molecules, developed in the 80s and 90s.
Methylation-sensitive restriction endonucleases, pioneered by Adrian Bird, have given way to the higher resolution attained through sodium bisulphite conversion. Tost touches on ways of assaying global methylation levels through HPLC methods and methyltransferase digestion. The latter uses radiolabeled molecules and may be increasingly less attractive to alternatives, such as new pyrosequencing based bioluminometric detection assays. Other genome-wide valuations include microarray-based technologies, utilizing either BAC clones or small length oligonucleotide arrays. One downside to these approaches is the reliance on PCR enrichment.
In fact, reliance on PCR and enzymatic digestion are hallmarks of various global DNA methylation experiments, including differential methylation hybridization arrays and methylation-sensitive arbitrary primed PCR methods. Methylated DNA immunoprecipitation does often require PCR but is not reliant on methyl-sensitive enzymes. Instead it captures the methylated fraction of the genome via affinity of methyl-CpG-binding domain proteins. Tost discusses the various permutations of immunoprecipitation methods and their pros and cons.
One of the final chapter sections focuses on sequencing methods; the earliest and still very popular Sanger sequencing of bisulphite converted DNA and quantitatively improved pyrosequencing technology. Unfortunately, there is no discussion of the growing popularity of Next-Generation Sequencing methods as alternatives to microarray. However, Tost provides a comprehensive method’s description for seemingly every DNA methylation question out there, making this particular chapter a valuable reference guide for academic and industry, and clinical and basic research labs alike.
You can find and purchase full copies of the Epigenetics at the Horizon Press website.