One of the most significant challenges in PCR design of bisulfite-converted genomic DNA is the design and optimization of primers to reduce PCR bias. When aiming to preserve the quantitative ratio of methylated-to-unmethylated CpG sites for quantitative methylation, it is imperative that the primer design be un-biased so that it will not select for one type of fragment over another.
The nature of methylation analysis is that methylated and unmethylated DNA molecules will be amplified with greatly differing efficiencies. This is due to secondary structure formation (primer loops and dimers, template loops, mispriming) and long strings of T’s and G’s and C/T variation at CpG sites. Primers situated on CpG sites can selectively amplify either the methylated or unmethylated fragment, possibly producing bias in the final methylation analysis.
EpigenDx has expertise in PCR and assay design, and we validate the assay conditions using quantitative Pyrosequencing methylation technology to ensure methylated and unmethylated DNA are amplified at the same efficiency. We provide complete primer sets and assay conditions to our customers, optimized for bisulfite sequencing and other DNA methylation applications such as COBRA and HRM real-time PCR.
Search Our Pre-Validated Assays:
- Custom Primer Set, 100 Reactions*
- Assay Conditions for PCR amplification
|Validated Methylated Analysis Primer Set, 300 reactions
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*Note: Either the forward or reverse primer can be biotinylated.