Automated Validation of Polymerase Chain Reactions Using Amplicon Melting Curves

PCR, the polymerase chain reaction, is a fundamental tool of molecular biology. Quantitative PCR is the goldstandard methodology for determination of DNA copy numbers, quantitating transcription, and numerous other applications. A major barrier to large-scale application of PCR for quantitative genomic analyses is the current requirement for manual validation of individual PCR reactions to ensure generation of a single product. This typically requires visual inspection either of gel electrophoreses or temperature dissociation (“melting”) curves of individual PCR reactions—a time-consuming and costly process. Here we describe a robust computational solution to this fundamental problem. Using a training set of 10,080 reactions comprising multiple quantitative PCR reactions from each of 1,728 unique human genomic amplicons, we developed a support vector machine classifier capable of discriminating single-product PCR reactions with better than 99% accuracy. This approach has broad ...

• Type: Conference
• Year: 2005
• Where: CSB
• Authors: Tobias P. Mann, Richard Humbert, John A. Stamatoyannopoulos, William Stafford Noble
• Keywords: Bioinformatics, CSB 2005, Individual Pcr, PCR Reactions, Quantitative Pcr