A highly sensitive G-quadruplex-based luminescent switch-on probe for the detection of polymerase 3'-5' proofreading activity

Methods. 2013 Dec 15;64(3):224-8. doi: 10.1016/j.ymeth.2013.05.017. Epub 2013 Jun 2.

Abstract

We report herein a luminescent switch-on label-free G-quadruplex-based assay for the rapid and sensitive detection of polymerase proofreading activity using a novel iridium(III) complex as a G-quadruplex-selective probe. The interaction of the iridium(III) complex with the G-quadruplex motif facilitates the highly sensitive switch-on detection of polymerase proofreading activity. Using T4 DNA polymerase (T4 pol) as a model enzyme, the assay achieved high sensitivity and selectivity for T4 pol over other tested enzymes.

Keywords: ACN; Exo I; Exo III; G-quadruplex; Iridium(III) complex; Luminescent; PCR; Polymerase; Proofreading; T4 DNA polymerase; T4 pol; T7 Exo; T7 exonuclease; Taq DNA polymerase; Taq pol; acetonitrile; calf-thymus DNA; ctDNA; dAMP; dGMP; dNTP; deoxyadenosine monophosphate; deoxyguanosine monophosphate; deoxyribonucleoside triphosphate; double-stranded DNA; dsDNA; exonuclease I; exonuclease III; polymerase chain reaction; single-stranded DNA; ssDNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Biosensing Techniques
  • Coordination Complexes / chemistry*
  • DNA Probes / chemistry
  • DNA, Single-Stranded / chemistry
  • DNA-Directed DNA Polymerase / chemistry*
  • Enzyme Assays*
  • Exonucleases / chemistry
  • G-Quadruplexes
  • Iridium / chemistry
  • Luminescent Agents / chemistry*
  • Luminescent Measurements
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Sensitivity and Specificity

Substances

  • Coordination Complexes
  • DNA Probes
  • DNA, Single-Stranded
  • Luminescent Agents
  • Iridium
  • DNA-Directed DNA Polymerase
  • Exonucleases