Abstract
Identification of point mutations has been facilitated by a number of techniques, including transfection assays, oligonucleotide hybridization, electrophoretic migration of heteroduplexes, RNase mismatch analysis, direct sequencing, and DNA-polymerase catalyzed amplification. The large number of available techniques emphasizes the importance of developing rapid and reliable methods to identify molecular changes in genes. To date, we have concentrated on exploiting DNA-polymerase catalyzed amplification methods (1,2) in conjunction with direct manual and automated DNA sequencing to detect point mutations in the dihydrofolate reductase (DHFR) gene of methotrexate-resistant cells.
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References
Saiki, R. K., Scharf, S., and Faloona, F. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335.
Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.
Morandi, C., Masters, J., Mottes, M., and Attardi, G. (1982) Multiple forms of human dihydrofolate reductase messenger RNA. Cloning and expression in Escherichia coli of their DNA coding sequence. J. Mol. Biol. 156, 583–607.
McBride, L. J., Koepf, S. M., Gibbs, R. A., Salser, W., Mayrand, P. E., Hunkapiller, M. W., and Kronick, M. N. (1989) Automated DNA sequencing methods involving polymerase chain reaction. Clin. Chem. 35, 2196–2201.
Gyllensten, U. B. and Erlich, H. A. (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLS-DQA locus. Proc. Natl. Acad. Sci. USA 85, 7652–6765.
Tabor, S. and Richardson, C. C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767–4771.
Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237–238.
Dicker, A. P., Volkenandt, M., and Bertino, J. R. (1989) Detection of a single base mutation in the human dihydrofolate reductase (DHFR) gene from a methotrexate resistant cell line using the polymerase chain reaction (PCR). Cancer Commun. 1, 7–12.
Dicker, A. P., Volkenandt, M., Adamo, A., Barreda, C., and Bertino, J. R. (1989) Sequence analysis of a human gene responsible for drug resistance. A rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction. Bio Techniques 7, 830–838.
Heiner, C., Mallick, S., Wan, A., and Hunkapiller, M. (1988) Taq polymerase: increased enzyme versatility in DNA sequencing. Applied Biosystems, Inc. Sequencer Model 370 User Bulletin.
Tindall, K. R. and Kunkel, T. A. (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27, 6008–6013.
Cooper, D. N. and Krawczak, M. (1990) The mutational spectrum of single base-pair substitutions causing human genetic disease: patterns and predictions. Hum. Genet. 85, 55–74.
Keohavong, P. and Thilly, W. G. (1989) Fidelity of DNA polymerases in DNA amplification. Proc. Natl. Acad. Sci. USA 86, 9253–9257.
Krawczak, M., Reiss, J., Schmidtke, J., and Rosler, U. (1989) Polymerase chain reaction: replication errors and reliability of gene diagnosis. Nucleic Acids Res. 17, 2197–2201.
Rollo, F., Amici, A., Salvi, R., and Garbuglia, A. (1988) Short but faithful pieces of ancient DNA. Nature 335, 774.
Paabo, S. and Wilson, A. C. (1988) Polymerase chain reaction reveals cloning artifacts. Nature 334, 387,388.
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© 1993 Humana Press Inc., Totowa, NJ
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Dicker, A.P., Volkenandt, M., Bertino, J.R. (1993). Manual and Automated Direct Sequencing of Product Generated by the Polymerase Chain Reaction. In: White, B.A. (eds) PCR Protocols. Methods in Molecular Biology, vol 15. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-244-2:143
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DOI: https://doi.org/10.1385/0-89603-244-2:143
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-244-6
Online ISBN: 978-1-59259-502-0
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