LightCycler® FastStart DNA Master SYBR Green I

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

LORD-Q assay
Real-time PCR analysis was carried out on the LightCycler 480 II system (Roche) in 96-well plates. The PCR amplification was monitored by real-time measurement of the intercalation of the saturating fluorescent dye ResoLight (Roche) into dsDNA employing KAPA2G Fast Hot Start ReadyMix kit (Peqlab). For each nuclear or mitochondrial genomic region, HPLC-purified oligonucleotides (Sigma-Aldrich) were employed to detect DNA damage in long (3–4 kb) probes, while nested short (40–70 bp) amplicons served as internal undamaged reference.

For the LORD-Q method, we selected oligonucleotides meeting all required standards in PCR efficiency and specificity (11). Primers used for amplification of the mitochondrial and nuclear loci are listed in Supplementary Table S3. To exclude accidental co-amplification of nuclear mitochondrial sequences (numts) (12), we used validated mtDNA primers that do not anneal to any numts DNA present in the genome database. All primers, including mtDNA probe primers, yielded a single PCR product, as confirmed by agarose gel electrophoresis. The PCR efficiencies of all amplicons were calculated using standard template dilution series of 50, 25, 12.5 and 6.25 ng whole-cell DNA per reaction (Supplementary Figure S1A). The rtPCR reaction mix consisted of 1× KAPA2G Fast Hot Start ReadyMix, 0.05× LightCycler 480 ResoLight dye, 500 nM of each forward (sense) and reverse (antisense) primer for the long and short amplicon, and 50 ng of template DNA in a total volume of 20 µl per well. Cycling conditions were as follows: a pre-incubation phase of 5 min at 95°C was followed by up to 50 cycles of 10 s at 95°C, 10 s at 60°C and 1 s at 72°C (small amplicons or synthetic single-stranded oligonucleotide templates carrying a modified nucleotide) or 2:15 min at 72°C (large amplicons). To compare the accuracy and efficacy of the LORD-Q method with an established semi-long-run rtPCR-based approach, we employed a previously published protocol (8) using SYBR Green, Taq polymerase and established oligonucleotides to amplify mtDNA fragments of a maximum length of 974 bp.



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Manufacturer protocol

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