Sensitive detection of formamidopyrimidine-DNA glycosylase activity based on target-induced self-primed rolling circle amplification and magnetic nanoprobes.

We developed a novel approach to determine formamidopyrimidine DNA glycosylase (FPG) activity by taking advantage of target-induced self-primed rolling circle amplification (RCA) and magnetic nanoprobes. Herein, a unique nick (8-oxoguanine, 8-oxoG) was positioned in duplex DNA containing P-circle and P1, which together serve as a FPG substrate, RCA template, and RCA primer probe. The presence of FPG specifically binds 8-oxoG and cleaves the P-circle into two parts, producing 5'-phosphoryl termini. A phosphodiester bond between the 5'-phosphoryl and 3'-hydroxyl termini was formed with the addition of T4 DNA ligase, producing an unnicked circular strand. Using the unnicked strand as the RCA template, the P1 hybridized with the circle probe as a primer will trigger the RCA process. The RCA reaction produces amounts of long tandem-repeat DNA tiles with multiple recognizing regions for the FAM modified DNA probes (FP) and biotin-modified DNA probes (BP). With the streptavidin-biotin interaction, the BPs and FPs can be easily immobilized on the surface of streptavidin-modified magnetic microbeads (MBs). Due to the RCA enhanced and highly-concentrated fluorescence accumulation on the MBs, an ultralow detection limit of 1.033 U mL-1 for FPG was obtained. Combined with the high tolerance capability of human blood serum owing to magnetic isolation, the FPG assays in human blood serum were also obtained using fluorescence and confocal laser scanning microscopy. These results indicate that this robust self-primed RCA combined with magnetic nanoprobes is an excellent candidate for quantitatively monitoring the FPG activity responsible for DNA oxidative damage-related clinical diagnosis and therapy.

Inter-laboratory variation in DNA damage using a standard comet assay protocol.

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.