Abstract
The sequencing of DNA has undergone rapid improvement since the introduction of the chain-termination DNA sequencing method (1) and the construction of convenient single-stranded DNA cloning vectors, such as the bacteriophage M13 cloning vectors and their derivatives (2,3). The chain-termination method involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded DNA template. Synthesis is initiated at only one site where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by the incorporation of a nucleotide analog that will not support continued DNA elongation. The chain-terminating nucleotide analogs are the 2′, 3′-dideoxynucleotide 5′-triphosphates (ddNTPs), which lack the 3′-OH group necessary for DNA chain elongation. When proper mixtures of deoxynucleotide triphosphates (dNTPs) and one of the four ddNTPs are used, enzymecatalyzed polymerization will be terminated in a fraction of the population of chains at each site where the ddNTP is incorporated. Four separate reactions, each with a different ddNTP, give complete sequence information.
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Sanger, F., Nicklen, S., and Coulson, A. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463–5467.
Messing, J. (1983) New M13 vectors for cloning. Methods Enzymol. 101,20–79.
Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.
Tomley, F. (1996) M13 phage growth and single-stranded DNA preparation, in Methods in Molecular Biology: Basic DNA and RNA Protocols, vol. 58, (Harwood, A. J., eds.), Humana Press, Totowa, NJ, pp. 359–362.
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© 2001 Humana Press Inc.
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Yu, Q. (2001). M13 Sequencing. In: Graham, C.A., Hill, A.J.M. (eds) DNA Sequencing Protocols. Methods in Molecular Biology™, vol 167. Humana Press. https://doi.org/10.1385/1-59259-113-2:033
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DOI: https://doi.org/10.1385/1-59259-113-2:033
Publisher Name: Humana Press
Print ISBN: 978-0-89603-716-8
Online ISBN: 978-1-59259-113-8
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