title: Liquid chromatography-mass spectrometry analysis of DNA polymerase reaction products.
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.
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Goutam
authors.initials: G
authors.lastname: Chowdhury
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D053719:Tandem Mass Spectrometry; D051981:Uracil-DNA Glycosidase / Q000737:chemistry
chemical_list: D017931:DNA Primers; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase; D051981:Uracil-DNA Glycosidase