Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates

Bioorg Med Chem Lett. 2016 Jan 15;26(2):288-291. doi: 10.1016/j.bmcl.2015.12.034. Epub 2015 Dec 11.

Abstract

A simple and elegant method for inhibition of non-templated nucleotide addition by DNA polymerases and for following DNA 3'-heterogeneity in enzymatic DNA synthesis by primer extension (PEX) is described. When template bearing ortho-twisted intercalating nucleic acid (ortho-TINA) at the 5'-end is used, non-templated nucleotide addition is reduced in both the A- and B-family DNA polymerases (KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-) and Taq). Formation of a single oligonucleotide product was observed with ortho-TINA modified template and KOD XL, KOD (exo-), Bst 2.0, Deep Vent (exo-) and Taq DNA polymerases. This approach can be applied to the synthesis of both unmodified and base-modified oligonucleotides.

Keywords: DNA polymerases; Nucleotide addition; Oligonucleotides; Primer extension; Twisted intercalating nucleic acid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotin / chemistry
  • DNA / chemistry
  • DNA / genetics*
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • Geobacillus stearothermophilus / enzymology
  • Intercalating Agents / chemistry*
  • Pyrenes / chemistry*
  • Pyrococcus
  • Thermococcus / enzymology
  • Thermus / enzymology
  • Triphenylmethyl Compounds / chemistry

Substances

  • Intercalating Agents
  • Pyrenes
  • Triphenylmethyl Compounds
  • Biotin
  • DNA
  • DNA-Directed DNA Polymerase