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Unmodified ddNTPs for Sequencing & SNP Genotyping

Dideoxynucleotide triphosphates (ddNTPs) lack the 3'-OH group of dNTPs that is essential for polymerase-mediated strand elongation in a PCR. Therefore, ddNTPs are used in combination with a modified Taq polymerase (e.g. JBS Sequencing polymerase or Thermosequenase™) as 3'-end chain terminators in Sanger sequencing[1] (Fig. 1) and single nucleotide polymorphisms (SNPs) genotyping by single base pair extension (SBE)[2] (Fig. 2). Our ddNTPs are > 98 % pure (HPLC) and functionally tested in chain termination sequencing.


Figure 1: The enzymatic dideoxy chain termination sequencing method of Sanger[1] 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).

Figure 2: The single basepair extension (SBE) assay is a reliable method for the detection of a priori known locations of single nucleotide polymorphisms (SNPs)[2]. It relies on the extension of a primer, designed to bind one nucleotide upstream of the polymorphic spot, by a matching ddNTP. Subsequent detection of the incorporated ddNTP is performed by mass spectrometry of the extended primer, thus revealing the nucleotide base at that position on the template strand[3].

Selected References

[1] Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463.
[2] Syvänen et al. (1999) From gels to chips: Minisequencing primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum. Mut. 13:10.
[3] Griffin et al. (2000) Single nucleotide polymorphism analysis by MALDI-TOF mass spectrometry. Trends Biotechnol. 18 (2):77.