Automated validation of polymerase chain reaction amplicon melting curves.
The polymerase chain reaction (PCR) 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 PCRs to ensure generation of a single product. This typically requires visual inspection either of gel electrophoreses or temperature dissociation ("melting") curves of individual PCRs--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 PCRs from each of 1728 unique human genomic amplicons, we developed a support vector machine classifier capable of discriminating single-product PCRs 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.
Mann
Tobias P
TP
Department of Genome Sciences, University of Washington, Seattle, WA, USA. mann@gs.washington.edu
Humbert
Richard
R
Stamatoyannopolous
John A
JA
Noble
William Stafford
WS
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*; D044366:Transition Temperature
D004247:DNA