Accurate methods to quantitate the methylation levels of samples are required, mostly to analyze tumor clinical samples, for large- scale studies. The authors here report a method called Combined Bisulfite Restriction Analysis (COBRA), which fulfills the above mentioned requirements and also is compatible with paraffin samples. The method consist in (1) carrying out a bisulfite modification; (2) then a PCR amplification; (3) doing a digestion with restriction enzymes whose sites would be different between experimental and control samples; and finally (4) a hybridization with a later phosphorimager quantitation.Clearly, the method relies on the distinction of restriction sensitive sites, and the fraction that contains CpG sites will be a reflection of the percentage of methylation in the original sample. The authors tested the reliability of this development with the human estrogen receptor gene, that is increasingly methylated in some colorectal cancer forms. Finally, Xiong and Laird discussed that this method overcomes problems in using hybridizations alone (that are excellent for quantitation but not compatible with paraffin samples), in restriction digestion followed by PCR (less sensitivity), and in MSP (a technique that is only qualitative). Comment: This method could have been a great development by the time of the publication of this paper, but currently it is not widely used, perhaps because it relies on restriction enzyme digestions (that depend on the quality of the sample, and other crucial factors), and in Southern hybridizations (a non- high- throughput method with many problems). Developments in mass spectrometry, capillary electrophoresis, real time PCR and pyrosequencing should lead to better ways to quantify methylation in samples, towards more precise cancer prognosis.
Z.Xiong and P.W.Laird. 1997. Nucleic Acids Res 25(12):2532-2534
epigenetics
Z.Xiong and P.W.Laird. 1997. Nucleic Acids Res 25(12):2532-2534
epigenetics
