Bisulfite Conversion, Meet COBRA

Workflow for the first steps of Combined Bisulfite Restriction Analysis. Image from Wikimedia Commons: Attribution under Creative Commons License:
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.

In 1997, Xiong and Laird provided this quantitative technique in a paper in Nucleic Acids Research that allowed them to analyze the DNA methylation patterns in tumor-suppressor genes from DNA from FFPE tissue. Briefly, DNA was purified from the FFPE microsections and treated with sodium bisulfite to convert any unmethylated cytosines to uracil. Methylated cytosines were not converted. The converted samples were desalted using a DNA clean-up system. The group then performed methylation insensitive PCR around the loci of interested, with PCR primers that did not hybridize in CpG regions. Essentially, this ensured that, regardless of conversion, the fragment of interest would be amplified. Post-amplification, the samples were again cleaned up and were then digested with a methylation sensitive restriction enzyme such as BstUI  or TaqI. These enzymes cleave at CpG sites and, if the starting sample was methylated, the cut site was retained. If the starting sample was not methylated, the bisulfite conversion and subsequent PCR would have changed any C’s to T’s (with a U intermediate following the conversion), and the cut site would be lost. Xiong and Laird then ran digested PCR product on an 8% denaturing polyacrylamide gel and transferred the resolved sample to Zetabind charged membrane via electroblotting. To visualize the bands quantitatively, the membranes were hybridized with 5’-end labeled oligos and quantitated using a phosphoimager.1

This technique allowed for the calculation of the percent methylation of the loci of interest. Other groups had used the Southern Blot technique to achieve this quantitation, but this was not feasible for samples from FFPE tissue because the amount and quality of purified DNA was a limiting factor. The COBRA method allowed scientists to study DNA methylation patterns in FFPE tissue in a quantitative manner.

Coming up next…..

Next, we’ll delve into approaches to study DNA methylation patterns on a global level. Stay tuned!

  1.  Xiong, X. and Laird, P.W.  1997.  COBRA: a sensitive and quantitative DNA methylation assay.  Nuc. Acids Res. 25:12, 2532-2534.
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