Chapter

Standardization and Quality Assurance in Fluorescence Measurements II

Volume 6 of the series Springer Series on Fluorescence pp 489-508

Date:

Quantitative Real-Time PCR: Fluorescent Probe Options and Issues

  • Marcia J. HoldenAffiliated withNational Institute of Standards and Technology, Biochemical Science Division Email author 
  • , Lili WangAffiliated withNational Institute of Standards and Technology, Biochemical Science Division

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Abstract

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.

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FRET Hybridization probe Molecular beacon Real-time quantitative PCR Scorpion primer SYBR Green I TaqMan probe