A homogeneous fluorescence assay for rapid and sensitive quantification of the global level of abasic sites in genomic DNA

Abstract

The apurinic/apyrimidinic (AP) sites are frequent DNA lesions in genomic DNA (gDNA). Here we report a facile approach for rapid quantification of the AP sites in gDNA with high selectivity and sensitivity. With the assistance of T4 pyrimidine dimer glycosylase, we covalently labeled the AP sites with 5'-hydroxylamine-modified oligonucleotide strand with high chemical selectivity against to naturally occurring formylated-bases, such as 5-formylcytosine and 5-formyluracil. Next, we sequentially removed the excessive labeling strands and triggered a signal amplification reaction with the labeled strands in a homogeneous system by flexible variation of the 3' or 5' terminal bases of an assistant strand and a fluorescent probe in the presence of a versatile exonuclease (lambda exonuclease). The detection of AP sites in gDNA was realized with an input of gDNA less than 500 ng and a limit of detection down to 0.2 fmol. The method enabled quantification of AP sites in gDNA from both normal cells and cells exposed to external damaging agents, showing the variation of AP sites level along with damaging and repair processes. The work has also provided a useful strategy for the rapid detection of other targeted sites in gDNA in a homogeneous system.

Keywords: Abasic sites; Covalently labeling; DNA repair; Fluorescence assay; Genomic DNA.