Abstract 832: Comparison of single step to two step PCR for DNA methylation status determination utilizing a system that continuously contains PCR products in plastic consumables
Authors: Ao Wanyuan , Bosh Derek , Emery Dale , Parry Robert , Adey Nils 
Published: August 2019

Abstract
DNA methylation specific PCR is a commonly used method to determine DNA methylation status. However, a well-known risk for PCR, and particularly methylation specific PCR, is carryover contamination from amplification products that can result in false positive PCR results in subsequent tests. This risk can be mitigated using careful technique, facility design, and biochemical methods such as uracil incorporation and subsequent uracil N-glycosylase (UNG) negative selection. Unfortunately, careful technique is highly user dependent, facility design can be cumbersome and expensive, and uracil incorporation is effective for only one step of two-step (nested) PCR reactions and cannot be used with the DNA bisulfite treatment methods used for methylation specific PCR. Our company has developed a system that mitigates PCR product contamination by continuously containing PCR products in plastic consumables and can interface with existing laboratory robotics, real time thermocycling instruments, and cassette electrophoresis gels. Here we utilize this system to compare the sensitivity and specificity of two-step multiplex nested PCR vs. single-step PCR for DNA methylation status detection applications using both qPCR and gel electrophoresis analysis. Published methylation status specific PCR primers that target CpG nucleotides in promotor regions were used to PCR amplify commercially available fully methylated and fully unmethylated DNA. The results demonstrate that a multiplex preamplification using amplicons for the CHAD, GFI1, MX2, NEU1, VWCE genes followed by monoplex nested methylation specific PCRs resulted in a Limit Of Detection (LOD) of 1 to 10 DNA template copies whereas performing just the monoplex methylation specific PCR (no preamplification) resulted in a LOD of 10 to 300 DNA template copies. If DNA isolated from macro dissected FFPE tissue was utilized, positive signals were identified using multiplex nested PCR whereas no signals were identified using single-step PCR. When mixtures of methylated and unmethylated DNA were utilized along with the GSTP gene amplicons, two-step nested PCR using were able to distinguish as low as 1% unmethylated DNA or 2% methylated DNA in a background of the other form, whereas single-step PCR was able to distinguish just 10% unmethylated or 33% methylated DNA under the same conditions. Furthermore, no PCR product contamination was observed upon amplification of samples collected from the external portion of the device demonstrating the PCR products were well contained.
Citation Format: Wanyuan Ao, Derek Bosh, Dale Emery, Robert Parry, Nils Adey. Comparison of single step to two step PCR for DNA methylation status determination utilizing a system that continuously contains PCR products in plastic consumables [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 832.