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. 2007;35(4):1377-88.
doi: 10.1093/nar/gkm010. Epub 2007 Feb 5.

DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair

Affiliations

DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair

Jinsong Chen et al. Nucleic Acids Res. 2007.

Abstract

As a gold standard for quantification of starting amounts of nucleic acids, real-time PCR is increasingly used in quantitative analysis of mtDNA copy number in medical research. Using supercoiled plasmid DNA and mtDNA modified both in vitro and in cancer cells, we demonstrated that conformational changes in supercoiled DNA have profound influence on real-time PCR quantification. We showed that real-time PCR signal is a positive function of the relaxed forms (open circular and/or linear) rather than the supercoiled form of DNA, and that the conformation transitions mediated by DNA strand breaks are the main basis for sensitive detection of the relaxed DNA. This new finding was then used for sensitive detection of structure-mediated mtDNA damage and repair in stressed cancer cells, and for accurate quantification of total mtDNA copy number when all supercoiled DNA is converted into the relaxed forms using a prior heat-denaturation step. The new approach revealed a dynamic mtDNA response to oxidative stress in prostate cancer cells, which involves not only early structural damage and repair but also sustained copy number reduction induced by hydrogen peroxide. Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.

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Figures

Figure 1.
Figure 1.
The effect of supercoiling of plasmid DNA on real-time PCR. Supercoiled plasmid DNA, pBR322 was treated with EcoR1, N.BstNB1 and Topoisomerase1 to generate linear, nicked circular and closed circular forms, respectively, and analyzed using gel electrophoresis and quantitative PCR assays. (A) Distribution of enzymes’ cutting sites and DNA markers for real-time PCR (rt-PCR, black bars) and long PCR (dashed lines) assays. (B) Electrophoresis of treated plasmid DNA in 1% agarose gel. DNA bands were visualized by ethidium bromide staining after electrophoresis. (C) The effect of supercoiling on real-time PCR. Two short DNA markers (pBR102 and pBR1395) were analyzed for each samples using SYBR green dye. The relative amplification of real-time PCR was expressed as 2ΔCt. (D) Detection of blocking lesions using long PCR. Two long DNA markers (pBR-3901 bp and pBR-4068 bp) were amplified to detect the blocking effects of double and single strand breaks in plasmid DNA. The relative amplification of long PCR was expressed as AD/AO. The 3901-bp fragment excluded the EcoR1 site but flanked two nicking sites of N.BstNB1, while the 4068-bp fragment covered all three sites. Data from duplicate treatments were pooled and analyzed using the one-way analysis of variance in the Prism program (**P < 0.01).
Figure 2.
Figure 2.
Fe++-induced DNA strand breaks and structural disruption in plasmid DNA. Supercoiled pBR322 DNA was either digested with EcoR1, or treated for 15 min with various concentrations of Fe++ with or without mannitol in 100 mM potassium phosphate buffer (pH 7.0). (A) Electrophoresis of treated plasmid DNA in 1% agarose gel. Induced changes in conformational state of plasmid DNA were visualized by ethidium bromide staining after electrophoresis. The ratio of relaxed form (nicked circular + linear) vs. total DNA was computed using the Genetools software (Beacon House). (B) Detection of structural disruption in plasmid DNA induced by Fe++ and mannitol treatment using real-time PCR. (C) Detection of blocking lesions in plasmid DNA induced by Fe++ and mannitol treatment using long PCR. Data from duplicate treatments were pooled and analyzed using the one-way analysis of variance in the Prism program (*P < 0.05; **P < 0.01).
Figure 3.
Figure 3.
Fe++-induced structural damage in mtDNA in vitro. Total genomic DNA isolated from LNCaP cells was either digested by EcoR 1 or treated for 15 min with 10 and 100 μM Fe++ with or without mannitol in 100 mM potassium phosphate buffer (pH 7.0). (A) Detection of structural damage in mtDNA induced by Fe++ and mannitol treatment using real-time PCR. Two mtDNA markers (CO2 and Dloop) and two nuclear DNA markers (β-actin and β-globin) were analyzed using real-time PCR. The relative amplification of mtDNA markers and β-globin was calculated by normalizing to the reference gene of β-actin according to Equation I (see Materials and Methods). (B) Blocking effects of Fe++-induced DNA lesions in both mtDNA and nuclear DNA using long PCR. A 16.2-kb mtDNA and a 13.5-kb β-globin fragments were amplified using long PCR. The ratio of PCR amplification relative to the non-treated control was determined in both mtDNA and nuclear DNA. Data from duplicate treatments were pooled and analyzed using the one-way analysis of variance in the Prism program (*P < 0.05; **P < 0.01).
Figure 4.
Figure 4.
Effects of heat-denaturation of mtDNA on real-time PCR quantification. (A) Aliquots of 5 ng/μl of total genomic DNA isolated from LNCaP cells were heat-denatured at 95°C for different time periods and then used for real-time PCR amplification using two mtDNA markers and β-actin DNA marker. (B) Aliquots of 100 pg/μl of supercoiled pBR322 DNA were heat-denatured at 95°C and then analyzed using plasmid DNA marker pBR1395. The relative amplification of both mtDNA and plasmid DNA markers from heat-denatured templates was expressed as 2ΔCt, where ΔCt was Ctcontrol–Ctheated. (C) Electrophoresis of heat-denatured plasmid DNA in 1% agarose gel. Data from duplicate treatments were pooled. Differences between untreated and treated samples were all significant (P < 0.01).
Figure 5.
Figure 5.
Structural damage and repair in mtDNA of LNCaP cells induced by120 μM H2O2 treatment. LNCaP cells (2–2.5 ×ばつ 106) that seeded 48 h before experiment were treated with 120 μM H2O2 in serum-free medium for 15 and 60 min, respectively, to induce DNA damage. For recovery, LNCaP cells were first treated with 120 μM H2O2 for 60 min, then allowed to recover in fresh complete medium for 2 and 24 h, respectively. (A) Structural damage and repair in mtDNA of LNCaP cells. Two mtDNA (CO2 and Dloop) and two nuclear DNA (β-actin and β-globin) markers were used for real-time PCR using total genomic DNA isolated from each sample. The relative amplification of each marker was expressed as in Equation I with β-actin gene from each template serving as a reference. (B) Quantification of total mtDNA content in LNCaP cells using heat-denatured DNA templates. Aliquots of total genomic DNA from each sample were heat-denatured at 95°C for 6 min and then used for real-time PCR analysis using mtDNA marker CO2 (heated). The relative amplification of the marker was expressed as above with β-actin gene from each original template serving as a reference. Data from duplicate treatments were pooled and analyzed using the one-way analysis of variance in the Prism program (*P < 0.05; **P < 0.01).
Figure 6.
Figure 6.
Structural damage and copy number reduction in mtDNA of LNCaP cells induced by 240 μM H2O2 treatment. LNCaP cells were treated as in Figure 5 except that 240 μM H2O2 was used for both exposure and recovery treatments. (A) Structural damage in mtDNA of LNCaP cells induced by 240 μM H2O2. (B) Changes in total mtDNA content in LNCaP cells induced by 240 μM H2O2. (C) Preferential mtDNA damage in LNCaP cells detected using long PCR. A 16.2-kb mtDNA and 13.5-kb β-globin DNA were amplified using long PCR to detect blocking lesions induced in both fragments. The relative amplification of each marker was expressed as AD/AO. Data from duplicate treatments were pooled and analyzed using the one-way analysis of variance in the Prism program (*P < 0.05; **P < 0.01).
Figure 7.
Figure 7.
Model for simultaneous analysis of structural damage and repair as well as copy number change in stressed cells using real-time PCR. (A) Real-time PCR allows sensitive detection of relaxed (open circular/linear) or total mtDNA when all supercoiled DNA is converted into the relaxed forms using a heat-denaturation step. The ratio between relaxed and total mtDNA indicates the baseline levels of structural damage, while the quantitative differences between total and relaxed mtDNA represents the fraction of supercoiled DNA. (B) Induced structural damage and its subsequent repair can be detected by the increase or decrease of the relaxed fraction of mtDNA when total mtDNA content remains the same (I). However, quantitative changes in heat-denatured mtDNA indicate either reduction in mtDNA copy number due to fragmentation or increase due to nascent replication (II).

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