Molecular Biology: Real-time PCR

Molecular Biology:

Real-time PCR

Author: Dr Kgomotso Sibeko-Matjila

Licensed under a Creative Commons Attribution license.

Pre-requisites for the sub-module on Real-time PCR:

  1. General theory on Molecular Biology
  2. Polymerase chain reaction (PCR)

REFERENCES

  1. Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, P.C., Wolf, D.E., 1988.Detection of nucleic acid hybridization by nonradiative fluorescenceresonance energy transfer. Proc. Natl. Acad. Sci. 85 (23):8790–8794.
  2. Gibson, U.E., Heid, C.A., Williams, P.M.,1996. A novel method for real time quantitative RT‐PCR. Genome Res. 6:995-1001.
  3. He, L., Chinnery, P.F., Durham, S.E., et al., 2002. Detection and quantification of mitochondrialDNA deletions in individual cells by real‐time PCR. Nucleic Acids Research, 30:e68.
  4. Heid, C.A., Stevens, J., Livak, K.J., Williams, P.M.,1996. Real time quantitative PCR. Genome Res. 6:986-994.
  5. Holland, P.M., Abramson, R.D., Watson, R., Gelfand, D.H.,1991. Detection of specific polymerase chain reaction product by utilizing the 50–30 exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA, 88:7276-7280.
  6. Livak, K.J., Flood, S.J., Marmaro, J., Giusti, W., Deetz, K., 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4 (6): 357-362.
  7. Morrison, T.B., Weis, J.J., Wittwer, C.T.,1998. Quantification of low‐copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 24:954-962.
  8. Rutledge, R.G., Côté C., 2003. Mathematics of quantitative kinetic PCR and the application ofstandard curves. Nucleic Acids Res. 31:e93.
  9. Simon, A., Labalette, P., Ordinaire, I., et al. 2004. Use of fluorescence resonance energy transfer hybridization probes to evaluate quantitative real‐time PCR for diagnosis of ocular toxoplasmosis. J Clin Microbiol. 42:3681–3685.
  10. Ste´phane Swillens, Barbara Dessars, Hakim El Housni, 2008 Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data. Analytical Biochem. 373:370-376.
  11. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T., 2000. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 28 (19):3752–3761.
  12. Tyagi, S. and Kramer, F.R.,1996. Molecular beacons: Probes that fluoresce upon hybridization. Nat Biotechnol. 14:303-308.
  13. Tyagi, S., Bratu, D.P., Kramer, F.R.,1998. Multicolor molecular beacons for allele discrimination. Nat Biotechnol. 16:49-53.
  14. Vaerman, J.L., Saussoy, P., Ingargiola, I., 2004. Evaluation of real‐time PCR data. J. Biol. Regul. Homeost. Agents; 18:212–214.
  15. Whitcombe, D., Theaker, J., Guy, S.P., Brown, T., Little, S.,1999. Detection of PCR products using self‐probing amplicons and fluorescence. Nat Biotechnol. 17:804-807.
  16. Wittwer, C.T., Herrmann, M.G., Moss, A.A., Rasmussen, R.P.,1997a. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 22:130-138.
  17. Wittwer, C.T., Ririe, K.M., Andrew, R.V., David, D.A., Gundry, R.A., Balis, U.J.,1997b. The LightCycler: a microvolumemultisample fluorimeter withrapid temperature control. Biotechniques 22 (1), 176–181.

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