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Bisulfite PCR
CpG Island Methylation in Aging and Cancer  

Bisulfite-PCR for Restriction Analysis and/or Sequencing

Protocol written by Jean-Pierre Issa, based on several published papers.

 

Bisulfite-PCR followed by restriction is a rapid and semi-quantitative method of analyzing DNA methylation. The PCR products are also suitable for either direct sequencing or cloning and sequencing. The most important step here is primer selection.

Generally, we prefer to be as close to the transcription start site of the gene as possible. This is also dictated by the availability of restriction enzyme sites. Here is a general strategy:

Sequencing Bisulfite-PCR products gives you more information than restriction. You can  clone the PCR products (TA cloning, for example) and sequence multiple clones (automated sequencing works well here). Any C in the sequence reflects methylation (or, rarely, an artifact!). Alternatively, you can use direct sequencing: Design nested primers that include sequencing primers, PCR using the nested primers first, then sequence.

Good Luck


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