A New Edge in Bisulfite Conversion

methyledge_featureproduct_280x140I’ve talked quite a bit about bisulfite conversion and DNA methylation analysis in  past posts. Aberrant methylation events have significant impacts in terms of incidence of cancer and development disregulation. Researchers studying DNA methylation are often working with DNA from “difficult” tissues such as formalin-fixed, paraffin embedded tissues, which characteristically yield DNA that is more fragmented than that purified from fresh tissue. Traditional methods for bisulfite conversion involve a long protocol, harsh chemicals, and generally yield highly fragmented DNA. The DNA fragmentation may significantly impact the utility of the converted DNA in downstream applications such as bisulfite-specific PCR or bisulfite sequencing.

An ideal bisulfite conversion system would allow for complete conversion of a DNA sample in a short period of time, provide high yield of DNA, minimally fragment the DNA, work on a wide range of input DNA amounts (from a wide variety of sample types), and, while we’re at it, be easy to use and to store. Whew! That’s quite the list. Continue reading “A New Edge in Bisulfite Conversion”

Webinar Redux: DNA Methylation Mechanisms and Analysis Methods

Earlier this week I had an opportunity to participate in a webinar given by Dr. Karen Reece, an R & D scientist here at Promega Corporation. The title of the webinar: “DNA Methylation Mechanisms and Analysis Methods to Study this Key Epigenetic Control”.

Karen, a fellow blogger here at Promega Connections, gave an excellent presentation, and as you know, epigenetics is a hot topic these days. 

By definition, epigenetics is the ability of environmental factors to affect gene expression in a heritable manner. From Wikipedia: “In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence .” Continue reading “Webinar Redux: DNA Methylation Mechanisms and Analysis Methods”

Bisulfite Conversion and Next Gen Sequencing

WebinarsIn my last entry, I gave a little summary of one of many techniques that are used to study DNA methylation patterns in a loci-specific fashion using the COBRA technique. This time, we’ll take a look at a high-throughput, genome-wide method for analyzing DNA methylation status using a next generation sequencing approache called bisulfite sequencing, or Bis-Seq. Continue reading “Bisulfite Conversion and Next Gen Sequencing”

Bisulfite Conversion, Meet COBRA

Workflow for the first steps of Combined Bisulfite Restriction Analysis. Image from Wikimedia Commons: Attribution under Creative Commons License: http://en.wikipedia.org/wiki/File:Cobra_workflow.svg
Last time, I talked about the birth of the bisulfite conversion method for studying DNA methylation. Scientists exploited the fact that, when treated with sodium bisulfite, unmethylated cytosines undergo sulphonation and are converted to uracil. Methylated cytosines are unaffected by this treatment. This methylation-specific  alteration of the genome gave scientists a way to study the methylation patterns in DNA. One specific method for studying loci-specific DNA methylation patterns post-bisulfite conversion is called Combined Bisulfite Restriction Analysis, or COBRA. This method uses bisulfite conversion, followed by methylation sensitive restriction enzyme digestion to detect changes in DNA methylation patterns. Continue reading “Bisulfite Conversion, Meet COBRA”