CPD Course: REAL

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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 fluorescence resonance 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 mitochondrial DNA
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.
http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr/realtime-pcr-vs-traditional-pcr.html?ICID=EDI-Lrn2
7.
http://www.qiagen.com
8.
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.
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Molecular Biology: Real-time PCR
9.
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.
10. Rutledge, R.G., Côté C., 2003. Mathematics of quantitative kinetic PCR and the application of
standard curves. Nucleic Acids Res. 31:e93.
11. 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.
12. 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.
13. 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.
14. Tyagi, S. and Kramer, F.R., 1996. Molecular beacons: Probes that fluoresce upon hybridization. Nat
Biotechnol. 14:303-308.
15. Tyagi, S., Bratu, D.P., Kramer, F.R., 1998. Multicolor molecular beacons for allele discrimination. Nat
Biotechnol. 16:49-53.
16. Vaerman, J.L., Saussoy, P., Ingargiola, I., 2004. Evaluation of real‐time PCR data. J. Biol. Regul.
Homeost. Agents; 18:212–214.
17. 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.
18. 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.
19. Wittwer, C.T., Ririe, K.M., Andrew, R.V., David, D.A., Gundry, R.A., Balis, U.J., 1997b. The
LightCycler: a microvolumemultisample fluorimeter with rapid temperature control. Biotechniques 22
(1), 176–181.
20. www.appliedbiosystems.com
21. www.roche-applied-science.com/lightcycler/
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