|DNA methylation analysis using Single Nucleotide Primer Extension (SNuPE)
Single Nucleotide Primer Extension is a powerful method which can be used for the precise analysis of methylation in a certain position. The procedure is shown on the figure. You treat your DNA with bisulphite and then anneal a primer which ends immediately before the site of analysis. Then you perform two primer extension reactions, one with radiolabelled dCTP (ddCTP) and another with dTTP (ddTTP). In this reactions only one nucleotide will be added to the primer. Then you have to denature your reaction, run your products on acrylamide gel and analyse the activity of the labelled primer. This method can also be adapted for the use with fluorescence labelled nucleotides or terminators.
The primers for SNuPE should be defined to bisulphite treated sequence. If the primer contains a CG sequence you have to use (Y=C/T) at the position of C.
Protocol for 32P labelled nucleotides
|\METHODS\DNA methylation analysis\Single Nucleotide Primer Extension (SNuPE)
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