Nucleic acid detection and quantification are important clinical tools for infectious disease testing, genetic screening, and cancer research. The polymerase chain reaction (PCR) is often used to amplify specific sequences for analysis. The analysis of PCR products is currently performed after amplification is complete. We will develop a commercial instrument and techniques for analyzing PCR products during 10-20 minutes of amplification. Fluorescence will be used to follow hybridization during temperature cycling. The ability to monitor hybridization allows relative and absolute quantification of specific sequences and mutation detection. The melting and reannealing of PCR products can be followed fluorescently during PCR amplification. The product melting curve reflects product purity. Melting curves can be used for mutation detection and relative quantification. Analysis of PCR product reannealing can provide absolute product quantification. The hybridization of sequence specific probes can also be followed fluorescently. Probe melting curves can be used to detect mutations and assess relative quantification. Probe hybridization kinetics allow absolute sequence-specific quantification. These techniques do not require expensive probes or instrumentation. Automated fluorescence analysis during temperature cycling can provide clinically relevant answers in 10-20 minutes. Research productivity can also be enhanced by more efficient methods of amplification and analysis.
Idaho Technology is the only company that manufactures instrumentation for DNA amplification in 10-20 minutes. Using fluorescence to follow melting and hybridization during temperature cycling is a powerful approach for product identification, quantification, and mutation detection. The techniques and instrumentation developed should be widely used in academics, the biotechnology industry, and clinical medicine.
| Lyon, Elaine; Wittwer, Carl T (2009) LightCycler technology in molecular diagnostics. J Mol Diagn 11:93-101 |
