The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.
|Original language||English (US)|
|Journal||Journal of Visualized Experiments|
|State||Published - Mar 5 2018|
Bibliographical noteFunding Information:
This work was funded by the National Institutes of Health (ES023350). Lisa Chesner is supported by Training Grant 5T32HL007741. We thank Natalia Tretyakova (University of Minnesota) and Ashis Basu (University of Connecticut) and their lab members for support and technical advice during the early and intermediate stages of this work.
© 2018, Journal of Visualized Experiments. All rights reserved.
- DNA repair
- DNA-protein crosslinks
- Human oxoguanine glycosylase
- Issue 133
- Quantitative polymerase chain reaction
- Taq polymerase