Methyl Primer Express® Software and influence of amplicon characteristics to successrate in sequencing of bisulfite treated DNA
A well known method to study methylation patterns is to treat gDNA by sodium bisulfite to distinguish methylated cytosine (5mC) from unmethylated C, which is deaminated to uracil (U) and replaced by thymine (T) in subsequent amplification. 5mC still remains as C. Subsequent amplification can focus on selective amplification of methylation patterns in CpG islands (methyation specific PCR, MSP) or on amplification of bisulfite treated (converted) gDNA (Bisulfite treatment specific PCR, BIS). Selection of PCR focus is done by primer design. After PCR, sequencing can clarify the methylation pattern but several factors must be taken into account to ensure reliable data.
During the bisulfite treatment base composition will undergo dramatical changes. This must be taken under consideration during primer and amplicon design for the initial amplification. Dependend on the base composition of the target region the design may change the originally focused strategy for sequencing.
In first instance, strategy is depending on preferred outcome:
1. More or less methylated? / Which CpG in target region is differentially methylated? > Direct sequencing of PCR products.
2. Semi-quantitative results > Cloning of PCR products and sequencing of multiple clones.
In a second instance, the base composition of the amplicon itself will lead to the conclusion, whether direct sequencing of PCR products can be done or not.
Here we use a new PC Software called “Methyl Primer Express®” to design BIS oligos on different promotor-target regions to show examples for critical amplicons and recommendations for successful amplification and sequencing.