title: Automated validation of polymerase chain reactions using amplicon melting curves.
abstract: PCR, the polymerase chain reaction, is a fundamental tool of molecular biology. Quantitative PCR is the gold-standard 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 utility, and eliminates a major bottleneck to widespread application of PCR for high-throughput genomic applications.
authors.affiliation: Department of Genome Science, University of Washington, Seattle, WA, USA.
authors.forename: Tobias P
authors.initials: TP
authors.lastname: Mann
authors.forename: Richard
authors.initials: R
authors.lastname: Humbert
authors.forename: John A
authors.initials: JA
authors.lastname: Stamatoyannopolous
authors.forename: William Stafford
authors.initials: WS
authors.lastname: Noble
mesh_terms: D000465:Algorithms; D001185:Artificial Intelligence; D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D009691:Nucleic Acid Denaturation; D010363:Pattern Recognition, Automated / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D004247:DNA