Deoxyuridine Triphosphate (dUTP)-Dependent Sizing Variance of Polymerase Chain Reaction (PCR) Products on the Agilent 2100 Bioanalyzer System

To avoid false positive Polymerase Chain Reaction (PCR) detection, caused by cross-contamination with amplicons from earlier experiments, deoxyuridine triphosphate (dUTP) is commonly used as a substitute for deoxythymidine triphosphate (dTTP) in PCR mastermixes. This allows the performance of a decontamination step prior to PCR amplifi cation. Analysis of PCR products on the Agilent 2100 Bioanalyzer system shows that there are sizing differences depending on the nucleotide used in the mastermix. Introduction The use of dUTP in PCR mastermixes is critical for many applications, such as pathogen detection and GMO analysis. It enables the use of uracil-N-glycosidase (UNG) to remove any uridine-containing DNA from PCR reactions prior to amplifi cation, thus avoiding false positive detection due to contamination with amplicons from earlier runs. When the PCR products were analyzed using a DNA 1000 assay on a 2100 Bioanalyzer system, it was observed that the 2100 Expert Software showed different sizing depending upon the nucleotide used in the mastermix. Deoxyuridine Triphosphate (dUTP)-Dependent Sizing Variance of Polymerase Chain Reaction (PCR) Products on the Agilent 2100 Bioanalyzer System