What are the general recommendations for designing primers for bisulfite-treated/deaminated DNA?

We recommend designing long (~26-35) oligonucleotide primers to amplify bisulfite treated/deaminated DNA. Because bisulfite DNA is often damaged, it is most helpful to design targets between 150-500 bp. Note that since the DNA strands are no longer complementary after bisulfite-treatment/deamination, an individual primer set will only amplify one strand of the target sequence. The first primer should be designed to anneal to the converted target sequence. The second primer should be designed to anneal to the extension product of the first primer, not the opposite template strand, as would be the case in traditional PCR. Primers with higher annealing temperatures (>60°C as determined by the NEB Tm calculator) are recommended for optimal performance. If needed, include as many guanines as possible in the priming region or add additional bases to increase the Tm. Longer primers will also increase specificity. Ideally, CpG sites should be avoided; if essential, include them on the 5′-end of the primer and have them synthesized with a mixed base (Y= C/T, R= G/A) at the cytosine position. Computer programs such as MethPrimer, methBLAST or BiSearch can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.

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