Abstract
The advantages of direct sequencing of polymerase chain reaction (PCR) products over conventional sequencing of cloned, single-stranded DNA are manifold. Speed is perhaps the greatest asset. Time-consuming creation and screening of libraries, fragment purification, subcloning, bacterial transfection, and plasmid preparation steps are all eliminated. Cost is an advantage for similar reasons. The ability to sequence thousands or even millions of different templates at once, thereby obtaining pooled averages of mutated or polymorphic sequences, is another benefit. Moreover, short DNA sequences not obtainable by conventional cloning, such as DNA from paraffin-embedded tissues, can be sequenced using PCR sequencing.
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References
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 HLA-DQA locus. Proc. Natl. Acad. Sci. USA 85, 7652–7656.
Sarkar, G. and Sommer, S. S. (1988) RNA amplification with transcript sequencing (RAWTS). Nucleic Acids Res. 16, 5197.
Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. D. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436–9440.
Engelke, D. R., Hoener, P. A., and Collins, F. S. (1988) Direct sequencing of enzymatically amplified human genomic DNA. Proc. Natl. Acad. Sci. USA 85, 544–548.
Wrischnik, L. A., Higuchi, R. G., Stoneking, M., Erlich, H. A., Arnheim, N., and Wilson, A. C. (1987) Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA. Nucleic Acids Res. 15, 529–541.
Meltzer, S. J., Mane, S. M., Wood, P. K., Johnson, L., and Needleman, S. W. (1990) Sequencing products of the polymerase chain reaction directly, without purification. BioTechniques 8, 142–148.
Williams, J. F. (1989) Optimization strategies for the polymerase chain reaction. Biotechniques 7, 762–770.
Wahlberg, J., Lundeberg, J., Hultman, T., and Uhlen, M. (1990) General colo-rimetric method for DNA diagnostics allowing direct solid-phase sequencing of the positive samples. Proc. Natl. Acad. Sci. USA 87, 6569–6573.
Casanova, J.-L., Pannetier, C., Jaulin, C., and Kourilsky, P. (1990) Optimal conditions for directly sequencing double-stranded PCR products with Sequenase. Nucleic Acids Res. 18, 4028.
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© 1993 Humana Press Inc., Totowa, NJ
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Meltzer, S.J. (1993). Direct Sequencing of Polymerase Chain Reaction Products. In: White, B.A. (eds) PCR Protocols. Methods in Molecular Biology, vol 15. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-244-2:137
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DOI: https://doi.org/10.1385/0-89603-244-2:137
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-244-6
Online ISBN: 978-1-59259-502-0
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