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

Chen J, Kadlubar FF, Chen JZ - Nucleic Acids Res. (2007)

Bottom Line: 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.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.Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Division of Urology, McGill University Health Centre and Research Institute, Montreal, Quebec H3G 1A4, Canada.

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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Related in: MedlinePlus

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).
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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).

Mentions: The dramatic effect of conformational structure on real-time PCR rendered the technique useful in detecting structure-mediated DNA damage. Ferrous iron, a transition metal that promotes hydroxyl radical production (47), was used to introduce random DNA damage to plasmid DNA, while mannitol was used as a free radical scavenger to protect DNA from Fe++-induced hydroxyl radical attack. As visualized by gel electrophoresis (Figure 2A), Fe++ treatment alone induced a dose-dependent increase in percentage of the open circular form of plasmid DNA, indicating an accumulation of single-strand breaks. Addition of mannitol, however, dramatically reduced the formation of open circular DNA, which was consistent with the suppressing effect of hydroxyl radical scavengers on the formation of DNA strand breaks induced by transition metals (47,48). When Fe++-treated samples were analyzed using real-time PCR, the increase in real-time PCR amplification signal was positively correlated with the percentage of open circular DNA in each sample, with the maximum responses observed when most, if not all, of the supercoiled molecules were converted into the nicked circular forms as in 50 and 100 μM Fe++ treatments (Figure 2B). Thus, real-time PCR amplification increased as single-strand DNA breaks were introduced into an initially supercoiled DNA. Since neither the amplification signal (Figure 2B) nor the percentage of open circular DNA (Figure 2A) increased between 50 and 100 μM Fe++ treatments, the real-time PCR signal was more correlated with the percentage of molecules containing at least one strand break than with the number of breaks in each molecule. The accumulation of multiple DNA strand breaks in the same molecule appeared to have no additional impact on real-time PCR. On the other hand, long PCR analysis of the Fe++-treated DNA revealed a very different pattern of amplification with both increased and decreased PCR products observed, depending on the concentration of Fe++ treatments (Figure 2C). The complicated pattern of amplification signals suggests that randomly induced DNA strand breaks also have a 2-fold effect on long PCR amplification. Perhaps, the first strand break relaxes the supercoiling and increases the amplification signal by allowing complete separation of DNA strands. All breaks reduce amplification signal by blocking fork progression. The final result is complex kinetics of signal versus break. Thus, real-time PCR was more sensitive to detect and quantify structural disruption of supercoiled DNA induced by strand breaks, especially at the low levels.Figure 2.


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

Chen J, Kadlubar FF, Chen JZ - Nucleic Acids Res. (2007)

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).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851651&req=5

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).
Mentions: The dramatic effect of conformational structure on real-time PCR rendered the technique useful in detecting structure-mediated DNA damage. Ferrous iron, a transition metal that promotes hydroxyl radical production (47), was used to introduce random DNA damage to plasmid DNA, while mannitol was used as a free radical scavenger to protect DNA from Fe++-induced hydroxyl radical attack. As visualized by gel electrophoresis (Figure 2A), Fe++ treatment alone induced a dose-dependent increase in percentage of the open circular form of plasmid DNA, indicating an accumulation of single-strand breaks. Addition of mannitol, however, dramatically reduced the formation of open circular DNA, which was consistent with the suppressing effect of hydroxyl radical scavengers on the formation of DNA strand breaks induced by transition metals (47,48). When Fe++-treated samples were analyzed using real-time PCR, the increase in real-time PCR amplification signal was positively correlated with the percentage of open circular DNA in each sample, with the maximum responses observed when most, if not all, of the supercoiled molecules were converted into the nicked circular forms as in 50 and 100 μM Fe++ treatments (Figure 2B). Thus, real-time PCR amplification increased as single-strand DNA breaks were introduced into an initially supercoiled DNA. Since neither the amplification signal (Figure 2B) nor the percentage of open circular DNA (Figure 2A) increased between 50 and 100 μM Fe++ treatments, the real-time PCR signal was more correlated with the percentage of molecules containing at least one strand break than with the number of breaks in each molecule. The accumulation of multiple DNA strand breaks in the same molecule appeared to have no additional impact on real-time PCR. On the other hand, long PCR analysis of the Fe++-treated DNA revealed a very different pattern of amplification with both increased and decreased PCR products observed, depending on the concentration of Fe++ treatments (Figure 2C). The complicated pattern of amplification signals suggests that randomly induced DNA strand breaks also have a 2-fold effect on long PCR amplification. Perhaps, the first strand break relaxes the supercoiling and increases the amplification signal by allowing complete separation of DNA strands. All breaks reduce amplification signal by blocking fork progression. The final result is complex kinetics of signal versus break. Thus, real-time PCR was more sensitive to detect and quantify structural disruption of supercoiled DNA induced by strand breaks, especially at the low levels.Figure 2.

Bottom Line: 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.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.Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Division of Urology, McGill University Health Centre and Research Institute, Montreal, Quebec H3G 1A4, Canada.

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.

Show MeSH
Related in: MedlinePlus