Abstract
The PCR technique provides highly specific and sensitive means for analyzing nucleic acids, but it does not allow their direct quantification. This limitation originates from the fact that the efficiency of PCR depends on the amount of template sequence present in the sample, and the amplification is exponential only at low template concentrations (1). Because of this “plateau effect” of the PCR, the amount of the amplification product does not directly reflect the original amount of the template. Moreover, subtle differences in the reaction conditions, such as material from biological samples, might cause significant sample to sample variation in the final yield of the PCR product.
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References
Syvänen, A. C., Bengtström, M., Tenhunen, J., and Söderlund, H. (1988) Quantification of polymerase chain reaction products by affinity-based hybrid collection. Nucleic Acids Res. 16, 11,327–11,338.
Murphy, L. D., Herzog, C. E., Rudick, J. B., Fojo, A. T., and Bates, S. E. (1990) Use of polymerase chain reaction in the quantitation of mdr-1 gene expression, Biochemistry 29, 10,351–10,356.
Noonan, K. E., Beck, C., Holzmayer, T. A., Chin, J. E., Wunder, J. S., Andrulis, I. L., Gazdar, A. F., Willman, C. L., Griffith, B., von Hoff, D. D., and Roninson I. B. (1990) Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87, 7160–7164.
Chelly, J., Kaplan, J.-C., Maire, P., Gautron, S., and Kahn, A. (1988) Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 333, 858–860.
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.
Syvänen, A.-C., Aalto-Setälä, K., Harju, L., Kontula, K., and Söderlund, H. (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8, 684–692.
Syvänen, A.-C., Ikonen, E., Manninen, T., Bengtström, M., Söderlund, H., Aula, P., and Peltonen, L. (1992) Convenient and quantitative detection of the frequency of a mutant allele using solid-phase minisequencing: Application to aspartylglucosaminuria in Finland. Genomics 12, 590–595.
Ikonen, E., Manninen, T., Peltonen, L., and Syvänen, A.-C. (1992) Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing. PCR Methods Appl. 1, 234–240.
Syvänen, A.-C., Sajantila, A., and Lukka, M. (1993) Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing. Am. J. Hum. Genet. 52, 46–59.
Suomalainen, A., Kollmann, P., Octave, J.-N., Säderlund, H., and Syvänen, A.-C. (1993) Quantification of mitochondrial DNA carrying the tRNA8344Lys point mutation in myoclonus epilepsy and ragged-red-fiber disease, Eur. J. Hum. Genet. 1, 88–95.
Suomalainen, A., Majander, A., Pihko, H., Peltonen, L., and Syvänen, A.-C. (1993) Quantification of tRNA3243Leu point mutation of mitochondrial DNA in MELAS patients and its effects on mitochondrial transcription. Hum. Mol. Genet. 2, 525–534.
Laan, M., Grön-Virta, K., Salo, A., Aula, P., Peltonen, L., Palotie, A., and Syvänen, A.-C. (1995) Solid-phase minisequencing confirmed by FISH analysis in determination of gene copy number. Hum. Genet. 96, 275–280.
Syvänen, A.-C., and Säderlund, H. (1993) Quantification of polymerase chain reaction products by affinity-based collection. Meth. Enzymol. 218, 474–490.
Pastinen, T., Partanen, J., and Syvänen, A.-C. (1996) Multiplex, fluorescent solidphase minisequencing for efficient screening of DNA sequence variation. Clin. Chem. 42, 1391–1397.
Bengtström, M., Jungell-Nortamo, A., and Syvänen, A.-C. (1990) Biotinylation of oligonucleotides using a water soluble biotin ester. Nucleotides 9, 123–127.
Wu, R., Wu, N.-H., Hanna, Z., Georges, F., and Narang, S. (1984) Oligonucleotide Synthesis: A Practical Approach (Gait, M. J., (ed.), IRL Press, Oxford, UK.
Ihalainen, J., Siitari, H., Laine, S., Syvänen, A.-C., and Palotie, A. (1994) Towards automatic detection of point mutations: use of scintillating microplates in solid-phase minisequencing. BioTechniques 16, 938–943.
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© 1999 Humana Press Inc., Totowa, NJ
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Suomalainen, A., Syvänen, AC. (1999). Quantitative Analysis of Human DNA Sequences by PCR and Solid-Phase Minisequencing. In: Kochanowski, B., Reischl, U. (eds) Quantitative PCR Protocols. Methods in Molecular Medicine™, vol 26. Humana Press. https://doi.org/10.1385/0-89603-518-2:233
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DOI: https://doi.org/10.1385/0-89603-518-2:233
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