When a portrait artist is drawing a subject, it is often helpful to view the work in progress, as a reflection in a mirror. Somehow it re-attunes the mind’s eye to better assess the quality of the image. In a similar way, recently developed methods allow scientists to reflect on disease, or developmental states, by isolating NON-methylated DNA for profiling.
Illinngworth et al., cleverly developed the CAP method in 2008. It produces profiles of non-methylated DNA using a zinc finger CxxxC domain which specifically recognizes non-methylated CpGs. The downsides to this method are that it requires a high-resolution chromatographic system, a large amount of the recombinant CxxC module, large amounts of input DNA (to account for elution and handling steps), and it’s labor intensive.
There is now a simplified way to study your samples’ CG islands. See Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA Nucleic Acids Research (Dec. 10th, 2011) Blackledge et al. which explains how Bio-CAP adapts the CAP method to make it useful for “…very small quantities of DNA, and massively parallel sequencing-based genome-wide analysis of non-methylated DNA.”
Method notes from this publication include…
- Compare specific loci for changes in methylation status using small quantities of both the sample DNA and of high affinity CxxC domain NeutrAvidin bead elutions analyzed by qPCR.
- Resulting profiles of non-methylated and non-hydroxymethylated DNA, are nearly linear and proportional to CpG density.
- The method can be modified to use magnetic CxxC beads, reducing the procedure time to about two hours.
- The authors suggest that an automated purification system would probably work well for the magnetic Bio-CAP assay
- Genome-wide analysis by coupling to high throughput sequencing, allows for multiple samples analysis in parallel with vastly improved ability to view non-methylated DNA, when compared to MeDIP-seq data.
Blackledge, N., Long, H., Zhou, J., Kriaucionis, S., Patient, R., & Klose, R. (2011). Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA Nucleic Acids Research DOI: 10.1093/nar/gkr1207