How are the primers in the EpiTect Methyl qPCR Array designed?
The primers are designed around CpG islands known to be hypermethylated under relevant biological conditions or in relevant biological samples. Besides the usual specific requirements that real-time PCR primers must meet, the amplicon sequence must contain both restriction sites for both the methylation-sensitive and methylation-dependent enzymes. As a result, the amplicon lengths are longer than those seen for RT-PCR, which are around 150 to 400 bp. Our design algorithm also accounts for the GC-rich nature of the genomic DNA sequences that tend to make primer design more difficult, especially in and around CpG islands. Each primer pair is also experimentally verified at the bench for a single peak in the dissociation curve, and high amplification efficiencies