DNA Sequencing


DNA sequencing is the determination of base order in a DNA molecule. Methods for determining base order involve either chemical degradation or, more commonly, enzymatic synthesis of the region that is being sequenced. Automation of the DNA sequencing process is accelerating the progress of the Human Genome Project.

Keywords: human genome project; biotechnology; automated sequencing; gene

Figure 1.

Schematic view of Maxam–Gilbert reaction products. G, A, R, Y and C represent the specific chemical reactions that identify the relative positions of guanine (G), adenine (A), purine (‘R’; G and A), pyrimidine (‘Y’; C and T) and cytosine (C) bases, respectively. In this example the fragment is labelled at the 5′ end. Reading from the bottom towards the top of the gel, the banding pattern corresponds to the sequence 5′ GGTACGCCTGA 3′.

Figure 2.

Data produced using Sanger sequencing reaction. G, A, T and C represent the sequencing reaction products resulting from inclusion of ddGTP, ddATP, ddTTP or ddCTP. Since enzymatic synthesis proceeds 5′ to 3′, the smaller fragments identify bases that are closer to the primer (5′ end of the sequence information). (a) Schematic view of Sanger reaction products. The DNA sequence identified by this pattern of bands is indicated. (b) Photograph of corresponding sequence data.

Figure 3.

(a) Raw sequence data collected on an automated DNA sequencer (Perkin‐Elmer ABI PRISM Model 377). The four colours indicate the relative position of the bases in the DNA fragment. Each four‐colour vertical line corresponds to a different sequence reaction. The smaller fragments (nearer the cathode) identify bases that are closer to the primer (5′ end of the sequence information). (b) Portions of a representative, analysed sequence determined by the automated sequencer.


Further Reading

Ball S, Reeve MA, Robinson PS, Hill F, Brown DM and Loakes D (1998) The use of tailed octamer primers for cycle sequencing. Nucleic Acids Research 26: 5225–5227.

Burbelo PD and Iadarola MJ (1994) Rapid plasmid DNA sequencing with multiple octamer primers. BioTechniques 16: 645–650.

Collins FS, Patrinos A, Jordan E, Chakravarti A, Gesteland R, Walters L, the members of the DOE and NIH planning groups (1998) New goals for the US Human Genome Project: 1998–2003. Science 282: 682–689.

Hardin SH, Jones LB, Homayouni R and McCollum JC (1996) Octamer primed cycle sequencing: design of an optimal primer library. Genome Research 6: 545–550.

Jones LB and Hardin SH (1998a) Octamer‐primed cycle sequencing using dye‐terminator chemistry. Nucleic Acids Research 26: 2824–2826.

Jones LB and Hardin SH (1998b) Octamer sequencing technology: optimization using fluorescent chemistry. ABRF News 9 (2): 6–10.

Kieleczawa J, Dunn JJ and Studier FW (1992) DNA sequencing by primer walking with strings of contiguous hexamers. Science 258: 1787–1791.

Kotler LE, Zevin‐Sonkin D, Sobolev IA, Beskin AD and Ulanovsky LE (1993) DNA sequencing: modular primers assembled from a library of hexamers or pentamers. Proceedings of the National Academy of Sciences of the USA 90: 4241–4245.

Maxam AM and Gilbert W (1977) A new method for sequencing DNA. Proceedings of the National Academy of Sciences of the USA 74: 560–564.

Raja MC, Zevin‐Sonkin D, Shwartzburd J et al. (1997) DNA sequencing using differential extension with nucleotide subsets (DENS). Nucleic Acids Research 25: 800–805.

Sanger F, Nicklen S and Coulson AR (1977) DNA sequencing with chain‐terminating inhibitors. Proceedings of the National Academy of Sciences of the USA 74: 5463–5467.

Siemieniak DR and Slightom JL (1990) A library of 3342 useful nonamer primers for genome sequencing. Gene 96: 121–124.

Smith LM, Sanders JZ, Kaiser RJ et al. (1986) Fluorescence detection in automated DNA sequence analysis. Nature 321: 674–679.

Studier FW (1989) A strategy for high‐volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides. Proceedings of the National Academy of Sciences of the USA 86: 6917–6921.

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How to Cite close
Hardin, Susan H(Apr 2001) DNA Sequencing. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003147]