Nucleotides for Chain termination Sequencing

Dideoxynucleotide triphosphates (ddNTPs) lack the 3'-OH group of dNTPs that is essential for polymerase-mediated strand elongation in a PCR therefore leading to irreversible chain termination. Unmodified ddNTPs can be used in combination with a 5'-fluorescent labeled primer and a modified Taq polymerase (e.g. Thermosequenase™) for chain termination sequencing reactions ^[1,2] (Fig. 1).

Figure 1: The chain termination sequencing method with unmodified ddNTPs relies on the linear amplification of a single-stranded template DNA using a 5'-fluorescently labeled primer and a modified Taq polymerase. The synthesis of the complementary DNA strand starts at the specific priming site and ends with the incorporation of a chain-terminating ddNTP that is randomly introduced instead of its corresponding dNTP. By using the four different ddNTPs in four separate reaction vials, a set of extended primer strands terminated at each A, C, G, and T are obtained. When these fragments are separated on a suitable gel matrix the sequence information can be obtained from the migration order of the bands (bottom to top).

Selected References

[1] Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463.
[2] Smith et al. (1986) Fluorescence detection in automated DNA sequence analysis. Nature 321:674.