Abstract
The polymerase chain reaction (PCR) has revolutionized molecular biology. Portions of single-copy per cell genes (and cDNAs) prepared from very small tissue or cell samples can be specifically amplified for use in sequence determination, gene identification, and quantitation. Improvements to the method, such as the introduction of genetically engineered, thermostable polymerases, more precise thermocyclers and more efficient reverse transcriptases for mRNA conversion to cDNA, have combined to make RNA-PCR (also called reverse transcriptase, or RT-PCR) and PCR more reproducible and specific. Coupled with the high sensitivity of the reactions, RT-PCR and PCR are increasingly used as quantitative bio-analytical techniques.
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References
Murphy, L. D., Herzog, C. E., Rudick, J. B., Fojo, A. T., and Bates, S. E. (1990) Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29, 10,351–10,356.
Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 9717–9721.
Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 2725–2729.
Apostolakos, M. J., Schuermann, W. H. T., Frampton, M. W., Utell, M. J., and Willye, J. C. (1993) Measurement of gene expression by multiplex competitive polymerase chain reaction. Anal. Biochem. 213, 277–284.
Schwartz, H. E. and Ulfelder, K. J. (1992) Capillary electrophoresis with laser-induced fluorescence detection of PCR fragments using thiazole orange. Anal. Chem. 64, 1737–1740.
Guttman, A., Wanders, B., and Cooke, N. (1992) Enhanced separation of DNA restriction fragments by capillary gel electrophoresis using field strength gradients. Anal. Chem. 64, 2348–2351.
Landers, J. P., Oda, R. P., Spelsberg, J. A., Nolan, J. A., and Ulfelder, K. J. (1993) Capillary electrophoresis: a powerful microanalytical technique for biologically active molecules. Biotechniques 14, 98–111.
Dveksler, G. S., Basile, A. A., and Dieffenbach, C. W. (1992) Analysis of gene expression: use of oligonucleotide primers for glyceraldehyde-3-phosphate dehydrogenase. PCR Methods Appl. 1, 283–285.
Rye, H. S., Yue, S., Wemmer, D. E., Quesada, M. A., Haugland, R. P., Mathies, R. A., and Glazer, A. N. (1992) Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res. 20, 2803–2812.
Srinivasan, K., Morris, S. C., Girard, J. E., Kline, M. C., and Reeder, D. J. (1993) Enhanced detection of PCR products through use of TOTO and YOYO intercalating dyes with laser induced fluorescence-capillary electrophoresis. Appl. Theoretical Electrophoresis 3, 235–239.
Fasco, M. J., Treanor, C. P., Spivack, S., Figge, H. L., and Kaminsky, L. S. (1995) Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence. Anal. Biochem. 224, 140–147.
Fasco, M. J., Treanor, C., and Kaminsky, L. S. (1996) Cytochrome P450 mRNA induction: quantitation by RNA-polymerase chain reaction using capillary electrophoresis. Methods Enzymol. 272, 401–412.
Huang, Z. Q., Fasco, M. J., and Kaminsky, L. S. (1996) Optimization of DNase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR. Biotechniques 20, 1012–1020.
Fasco, M. J. (1997) Quantitation of estrogen receptor mRNA and its alternatively-spliced mRNAs in breast tumor cells and tissues. Anal. Biochem. 245, 167–178.
Forster, E. (1994) An improved general method to generate internal standards for competitive PCR. Biotechniques 16, 18–20.
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© 1999 Humana Press Inc., Totowa, NJ
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Fasco, M.J. (1999). Analysis of Amplified DNA Molecules by Capillary Electrophoresis and Laser Induced Fluorescence. In: Kochanowski, B., Reischl, U. (eds) Quantitative PCR Protocols. Methods in Molecular Medicine™, vol 26. Humana Press. https://doi.org/10.1385/0-89603-518-2:131
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DOI: https://doi.org/10.1385/0-89603-518-2:131
Publisher Name: Humana Press
Print ISBN: 978-0-89603-518-8
Online ISBN: 978-1-59259-262-3
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