3318 - What guidelines should I follow in designing primers for use with the EpiTect MSP Kit?

Several things should be taken into consideration when designing methylation-specific primers for use with the EpiTect MSP Kit:

 1.  Each primer should include at least one but preferably two CpG sites.

 2.  Both primers should have CpG sites at or near their 3’ end.

 3.  Primers should contain cytosines which are not in a CpG site.  This will make your primers

      specific for bisulfite-converted DNA since all unmethylated cytosines  will be converted to

      uracils in bisulfite-treated DNA.

4.  Primer pairs used for the detection of unmethylated and methylated DNA should cover the

     same CpG sites.  This is important to determine the methylation status of precisely defined

     CpG sites.

5.  Amplicons should have a length between 100 and 200 base pairs.