Abstract #1317: Primer design vs. PCR bias in methylation independent PCR amplifications (2009)

Most of the methodologies in methylation studies utilize primers that simultaneously amplify methylated and unmethylated alleles for post-PCR methylation status analyses. Methylated DNA strand after bisulfite modification has a different base content than unmethylated DNA one, with unchanged Cs on corresponding to the 5 methyl-C sites as opposed to Ts corresponding to at the C sites in unmethylated strand. Parallel amplification of two templates with different GC contents is biased towards the GC-poor templates (1,2) and therefore can lead to underestimation of methylation levels in screened material. We have used HRM (high resolution melting) to test the sensitivity of methylation detection of a panel of previously published primer sets (10 in total), which were designed to bind CpG free sequences. Our experiments showed that only half of the 10 primer sets was able to reproducibly detect 5% methylated templates from the mixes of methylated and unmethylated control DNAs. Overall, the sensitivity of the primers varied significantly with some primers detecting only 50% and others 5% of methylated sequence in unmethylated background. It was previously hypothesized that PCR bias occurs due to the presence of the secondary DNA structure of the template and can be overcome by increasing the annealing temperature of PCR amplification (1). We tested the influence of annealing temperature on PCR amplification. Nevertheless, increased annealing temperature of PCR amplification did not significantly improved sensitivity of above primers. We have re-addressed the issue of PCR bias in methylation experiments and developed a new primer design system, which relies on the inclusion of a limited number of CpG sites in the primer sequence 3. This allows for the control of the efficiency of primer binding to methylated/unmethylated templates by adjusting the annealing temperature and therefore allow the control of PCR bias during amplification (2). To date on the bases of new primer design system we have developed 30 Methylation Sensitive HRM assays. The sensitivity of our new assays is highly improved and ranges from 0.1% to 1% (4,5 and manuscripts in preparation). In summary the PCR bias when not controlled may compromise the specificity and sensitivity of the methylation screening assay and therefore has to be adequately addressed in the process of development of the assay. The above findings are of high importance for methyaltion markers research filed especially now when evaluation of single locus methyaltion markers in regard to their clinical application is performed in may laboratories all over the world. References: 1.%9P. M. Warnecke et al., Nucleic acids research 25 (21), 4422 (1997). 2.%9T. K. Wojdacz and L. L. Hansen, BioTechniques 41 (3), 274 (2006). 3.%9T. K. Wojdacz et al. BMC Res Notes 1, 54 (2008). 4.%9T.K. Wojdacz et al. Nature Protocols 3 (12) (2008). 5.%9T. K. Wojdacz and A. Dobrovic, Nucleic acids research 35 (6), e41 (2007). Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1317.