Liquid chromatography-mass spectrometry analysis of DNA polymerase reaction products

Curr Protoc Nucleic Acid Chem. 2011 Dec;Chapter 7:Unit 7.16.1-11. doi: 10.1002/0471142700.nc0716s47.

Abstract

This unit describes experimental and analytical procedures for characterizing the efficiency and fidelity of translesion DNA synthesis across various DNA damages by DNA polymerases in vitro. This procedure utilizes primer extension assays followed by LC-MS and LC-MS/MS analysis of the extension products. Detailed explanations for the analysis of the LC-MS/MS data for deciphering the nucleotide sequences of the DNA fragments are also presented. This approach provides a significant improvement over conventional methods, as it allows detection of misincorporation, as well as frameshift products.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • Chromatography, Liquid
  • DNA Primers / chemistry
  • DNA-Directed DNA Polymerase / chemistry*
  • Oligonucleotides / chemical synthesis
  • Oligonucleotides / chemistry
  • Sequence Analysis, DNA / methods*
  • Tandem Mass Spectrometry
  • Uracil-DNA Glycosidase / chemistry

Substances

  • DNA Primers
  • Oligonucleotides
  • DNA-Directed DNA Polymerase
  • Uracil-DNA Glycosidase