Quantitative Real-Time PCR: Fluorescent Probe Options and Issues

Fluorescence has played a vital role in the development of polymerase chain reaction (PCR)-based DNA amplification. In qualitative PCR, an end point reaction, the amplified DNA, is visualized using DNA intercalating fluorescent dyes. Creative uses of nucleotide probes with fluorescent tags have been developed for real-time quantitative PCR. These probes take advantage of the behavior and properties of fluorophores. There are advantages and disadvantages to various probe types as well as design considerations. Attention to these issues will help in the development of robust and accurate DNA quantification using real-time PCR. Open image in new window