Abstract
Kinetic or real-time PCR continues to develop at a rapid rate since its development in the early 1990s. New applications are continually being found for this technique and it is replacing conventional PCR in many fields because of its speed, reduced hands-on time, and because the closed-tube format greatly reduces the chance of reaction contamination. This chapter covers the basis of kinetic PCR and also discusses some of the parameters that need to be considered even before the actual amplification—such as nucleic acid extraction and the complementary DNA synthesis step required in the instance of gene expression studies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. (1992) Simultaneous amplification and detection of specific DNA sequences. BioTechnology (NY) 10, 413–417.
Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. BioTechnology (NY) 11, 1026–1030.
Livak, K. J. (1999) Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genetic Anal. 14, 143–149.
Lyon, E. (2001) Mutation detection using fluorescent hybridization probes and melting curve analysis. Expert Rev. Mol. Diagnostics 1, 92–101.
Hodgson, D. R., Clayton, S. J., Girdler, F., et al. (2001) ARMS allele-specific amplification-based detection of mutant p53 DNA and mRNA in tumors of the breast. Clin. Chem. 47, 774–778.
DeGraves, F. J., Gao, D. and Kaltenboeck, B. (2003) High-sensitivity quantitative PCR platform. Biotechniques 34, 106–115.
Espy, M. J., Rys, P. N., Wold, A. D., Uhl, J. R., Sloan, L. M., and Jenkins, G. D. (2001) Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using Isoquick, MagNA Pure and BioRobot 9604 methods. J. Clin. Microbiol. 39, 2233–2236.
Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15, 532–537.
Bustin, S. A. (2002) Quantification of mRNA using real-time reverse transcription real-time PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39.
Huang, Z., Fasco, M. J., and Kaminsky, L. S. (1996) Optimization of DNase I removal of contaminating DNA from RNA for use in quantitative RT-PCR. Biotechniques 20, 1012–1020.
Bauer, P., Rolfs, A., Regitz-Zagrosek, V., Hidebrandt, A., and Fleck, E. (1997) Use of manganese in RT-PCR reduces PCR artefacts resulting from DNase I digestion. Biotechniques 22, 1128–1132.
Hanaki, K., Nakatake, H., Yamamoto, K., Odawara, T., and Yoshikura, H. (2000) DNase I activity retained after heat inactivation in standard buffer. Biotechniques 29, 38–42.
Wiame, I., Remy, S., Swennen, R. and Sági, L. (2000) Irreversible heat inactivation of DNase I without RNA degradation. Biotechniques 29, 252–256.
Wilkening, S. and Bader, A. (2004) Quantitative real-time polymerase chain reaction: methodical analysis and mathematical model. J. Biomol. Tech. 15, 107–111.
Smith, C., Berg, D. Beaumont, S., Standley, N. T., Wells, D. N., and Pfeffer, P. L. (2006) Simultaneous gene quantification of multiple genes in individual bovine nuclear transfer blastocysts. Reproduction; Submitted.
Polimuri, S. K., Ruknudin, A. and Schulze, D. H. (2002) RNase H and its effects on PCR. Biotechniques 32, 1224–1225.
Lader, E. (2002) Ambion presentation.
Ståhlberg, A., Håkansson, J., Xian, X., Semb, H., and Kubista, M. (2004) Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 5, 509–515.
Lekanne Deprez, R. H., Fijnvandraat, A. C., Ruijter, A. M., and Moorman, A. F. M. (2003) Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal. Biochem. 307, 63–69.
Liss, B. (2002) Improved quantitative real-time RT-PCR for expression profiling of individual cells. Nucleic Acids Res. 30, e89.
Bromhead, C., Miller, J. H., and McDonald, F. J. (2006) Regulation of T-cadherin by hormones, glucocorticoid and EGF. Gene 374, 58–67.
Read, S. J. (2001) Recovery efficiencies of nucleic acid extraction kits as measured by quantitative LightCycler PCR. Mol. Pathol. 54, 86–90.
Bioinformatics, LLC. (2004) The market for real-time PCR reagents and instrumentation. http://www.gene2drug.com.
Sagner, G., Tabiti, K., Gutekunst, M., Soong, R., inventors. (2004) Method for the efficiency-corrected real-time quantification of nucleic acids. US Patent 6,691,041.
Tellmann, G. (2006) The E-Method: a highly accurate technique for gene-expression analysis. Nature Methods 3(7), ii.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Mackay, J. (2007). Introduction to Kinetic (Real-Time) PCR. In: Hilario, E., Mackay, J. (eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology, vol 353. Humana Press. https://doi.org/10.1385/1-59745-229-7:167
Download citation
DOI: https://doi.org/10.1385/1-59745-229-7:167
Publisher Name: Humana Press
Print ISBN: 978-1-58829-430-2
Online ISBN: 978-1-59745-229-8
eBook Packages: Springer Protocols