A recently published study has demonstrated the advantages of a modified real-time PCR-based technique for quantifying DNA damage.
The authors claimed the new method, called long-run real-time DNA-damage quantification (LORD-Q) is a significant advance over existing techniques in terms of sensitivity, cost, and ease of use, and can be used for applications such as determining cell type-specific DNA-damage frequencies, monitoring DNA repair capacities, or comparing DNA vulnerabilities of different genomic loci.
Researchers from the University of Tübingen and the German Cancer Research Center in Heildelberg, Germany, collaborated on the work, published last week in Nucleic Acids Research. They maintained the technique enabled "accurate sequence-specific quantification of DNA damage in [greater than] 3-kb probes for any mitochondrial or nuclear DNA sequence," according to the paper.
DNA lesions can lead to template amplification inversely proportional to the level of damage, making quantitative PCR-based approaches suitable. However, these approaches have the disadvantage of requiring labor-intensive determination of amplification efficiencies and normalization, have been limited to amplicon sizes of a few hundred basepairs, and use fluorescent dyes such as SYBR Green, which can inhibit the polymerase-driven elongation reaction by intercalating into double-stranded DNA.
The researchers also pointed out that standard protocols detect DNA damage "either in a global, sequence-independent manner, such as the single-cell gel electrophoresis (comet) assay and TUNEL staining, or are restricted to one or a few defined types of DNA lesions."
LORD-Q uses real-time PCR, but with a novel combination of reagents allowing for greater amplicon length. "After testing numerous PCR components such as DNA polymerases, DNA-intercalating dyes, and additives "the researchers wrote, they chose a combination of the high-fidelity and rapid KAPA2G Fast DNA polymerase from Kapa Biosystems and the second-generation fluorescent DNA dye ResoLight from Roche. "ResoLight, originally designed for high-resolution melting analyses, can be applied at very low concentrations and therefore leads to an only minor inhibition of template amplification," they wrote.
The authors used Jurkat cells in culture and four different methods to cause DNA damage: application of the genotoxins belomycin, cisplatin, or etoposide, or UV irradiation. Interestingly, they showed UV and cisplatin caused similar numbers of lesions in both nuclear and mitochondrial DNA, but bleomycin preferentially damaged mtDNA while etoposide was more damaging to nDNA.
The researchers then compared LORD-Q to conventional qPCR (using Taq polymerase and SYBR Green) as well as to a standard comet assay. LORD-Q was more sensitive to DNA damage caused by toxin concentrations of less than 10 nanomolar, compared to
the other methods.
They also tried their method on synthetic DNA oligonucleotides with different single-base modifications. LORD-Q was able to quantify "DNA-strand breaks, thymine dimers, abasic, and 5-hydroxymethylcytosine sites in an accurate and highly sensitive manner" — greater than or equal to 0.3 detectable lesions per 10 kb, they claimed.
Finally, to show some of the ways LORD-Q could be applied in biomedical research, the researchers used it to monitor DNA damage and repair in human induced pluripotent stem cells and dermal fibroblasts derived from the same background. They showed that LORD-Q could be used to report damage in two nDNA loci (the gene encoding OCT4 and the collagen 1 locus), and showed them to be differentially vulnerable to damage in stem cells or fully differentiated cells.
The authors have filed a patent application on the described method.