Overview of qPCR Molecular Probes

Andrei Laikhter1

1 Chemgenes Corporation, Wilmington, Massachusetts
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 10.5
DOI:  10.1002/9780470089941.et1005s11
Online Posting Date:  November, 2015
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In recent times, varieties of modified synthetic oligonucleotides conjugated with reporter molecules have been employed in hybridization‐based assays. These methods have broad applications in both basic molecular biology research and in clinical diagnostics and screening because they have high specificity to a target nucleic acid sequence. The development of the fluorescence resonance energy transfer (FRET) process has improved the performance and utility of these hybridization‐based assays. In this unit, we discuss the main principles of the design and synthesis of the qPCR probes. © 2015 by John Wiley & Sons, Inc.

Keywords: qPCR; fluorophore; quencher dye; molecular probes; molecular beacons; fluorescence resonance energy transfer (FRET)

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Table of Contents

  • Introduction
  • Fluorescence Spectra
  • qPCR Probe Design
  • Dark Quenchers Suitable for Ultrasensitive Probes
  • Examples of qPCR Probes and Commercial Availability
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
  Cook, R.M., Lyttle, M., and Dick, D. 2006. Dark quenchers for donor‐acceptor energy transfer. U.S. Patent 7,019,129.
  Frackman, S. Ekenberg, S., Hoffmann, K., Krenke, B., Sprecher, C., and Storts, D. 2005. Plexor technology: A new chemistry for real‐time PCR. Promega Notes 90:2‐4.
  Fraga, D., Meulia, T., and Fenster, S. 2014. Real‐time PCR. Curr. Protoc. Essen. Lab. Tech. 10:10.3:10.3.1‐10.3.40.
  Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5′—3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 88:7276‐7280. doi: 10.1073/pnas.88.16.7276.
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  Laikhter, A., Behlke, M., Walder, J.A., Roberts, K.W., and Yong, Y. 2008. Fluorescence quenching azo dyes, their methods of preparation and use. U.S. Patent 7,439,341.
  Laikhter, A., Walder, J.A., Behlke, M., and Podyminogin, M. 2010. Compounds and methods for labeling oligonucleotides. U.S. Patent 7,803,936.
  Laikhter, A., Srivastava, S., and Srivastava, N. 2011. Synthesis of novel azo‐dyes and their use in oligonucleotide synthesis. U.S. Patent 7,956,169.
  Landgraf, A., Reckmann, B., and Pingoud, A. 1991. Quantitative analysis of polymerase chain reaction (PCR) products using primers labeled with biotin and a fluorescent dye. Anal. Biochem. 193:231‐235. doi: 10.1016/0003‐2697(91)90014‐K.
  Lee, L.G., Connell, C.R., and Bloch, W. 1993. Allelic discrimination by nick‐translation PCR with fluorogenic probes. Nucleic Acids Res. 21:3761‐3766.doi: 10.1093/nar/21.16.3761.
  Nazarenko, I.A., Bhatnagar, S.K., and Hohman, R.J. 1997. A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res. 25:2516‐2521. doi: 10.1093/nar/25.12.2516.
  Sherrill, C.B., Marshall, D.J., Moser, M.J., Larsen, C.A., Daude‐Snow, L., Jurczyk, S., Shapiro, G., and Prudent, J.R. 2004. Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence. J. Am. Chem. Soc. 126:4550‐4556. doi: 10.1021/ja0315558.
  Tyagi, S. and Kramer, F.R. 1996. Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303‐308. doi: 10.1038/nbt0396‐303.
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