Let’s get ready to rumble! A new reagent, O-allylhydroxylamine, has entered the ring to challenge bisulfite conversion as the undisputed champion of DNA methylation analysis. In the latest issue of Nucleic Acids Research, chemists from Ludwig-Maximillians University in Germany introduced a new chemical reagent that could be a game-changer for distinguishing between cytosine (C) and methylcytosine (MeC) in a DNA sample.
O-allylhydroxylamine works like this: When DNA is treated with O-allylhydroxylamine, adducts are created with the cytosine molecules. These adducts can exist in two isomeric forms (E or Z). MeC adducts adopt the Z-form, which blocks polymerase activity, while C takes the E-form which reads as a C to T change during analysis. Although the readout (a C-to-T conversion) looks the same as with bisulfite, O-allylhydroxylamine gets there by generating different reaction products, instead of through changes in reactivity.
Aside from its hard-to-pronounce name, here are some of the other features that O-allylhydroxylamine brings to the battle:
- The reaction does not degrade sample DNA (Bisulfite treatment can destroy over 90% of DNA), so smaller input amounts can be used.
- Readouts and analysis methods (PCR amplification, pyrosequencing, etc.) are the same as bisulfite, meaning all of your favorite protocols can still be used.
- Reactions are sequence independent. You can study MeCs outside of CpGs, like in plant genomes.
- A luminescence assay has been developed detect relative amounts of MeC in a sample.
On paper, it seems that O-allylhydroxylamine has bisulfite’s best features but without all of the sample degradation. But, whether or not that will translate to the bench remains to be seen. In the meantime the authors continue to adapt hydroxylamines to work with other analysis methods like sequencing by synthesis.
See if you think that O-allylhydroxylamine is up for the challenge at Nucleic Acids Research, September 2010.