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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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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).
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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).

Mentions: Heat-denaturation, an essential step in both PCR amplification and real-time PCR, allows separation of two complementary DNA strands for specific primer binding. It could also introduce DNA strand breaks into supercoiled template DNA, leading to DNA degradation (49). To evaluate the effect of heat-denaturation on real-time PCR quantification of mtDNA, the same amounts of total genomic DNA from LNCaP cells were heat denatured at 95°C for increasing time periods before real-time PCR amplification. A significant increase in real-time PCR amplification was observed during the first 3 min denaturation before reaching a plateau after 4–6-min treatment (Figure 4A). Extended heat-denaturation over 6 min had no additional impact on real-time PCR of mtDNA. In contrast, β-actin DNA amplification exhibited little change within the first 6 min of treatment, but decreased slightly after 12 min (data not shown). The heat-denaturation effect on real-time PCR amplification was also observed in plasmid DNA and was accounted for by the accumulation of single-stranded molecules caused by strand breaks and denaturation (Figure 4B and C). Several practical implications could be derived from the effect of heat-denaturation on mtDNA quantification. For example, the initial heat-denaturing time in PCR amplification could have major impact on real-time PCR analysis of mtDNA content and structural changes. As demonstrated in both plasmid and mtDNA analyses earlier, real-time PCR was more efficient when acting upon relaxed from than upon supercoiled form DNA. Short initial denaturation time would better preserve the original ratios of supercoiled versus relaxed mtDNA, therefore enhancing the sensitivity for structural analysis. However, prolonged initial denaturation could convert all supercoiled DNA into its open-relaxed forms, eliminating structural effects all together. Incorporation of these two strategies could provide a simple new approach to analyze both relaxed form and total mtDNA content simultaneously using real-time PCR (see later).Figure 4.


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)

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

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

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).
Mentions: Heat-denaturation, an essential step in both PCR amplification and real-time PCR, allows separation of two complementary DNA strands for specific primer binding. It could also introduce DNA strand breaks into supercoiled template DNA, leading to DNA degradation (49). To evaluate the effect of heat-denaturation on real-time PCR quantification of mtDNA, the same amounts of total genomic DNA from LNCaP cells were heat denatured at 95°C for increasing time periods before real-time PCR amplification. A significant increase in real-time PCR amplification was observed during the first 3 min denaturation before reaching a plateau after 4–6-min treatment (Figure 4A). Extended heat-denaturation over 6 min had no additional impact on real-time PCR of mtDNA. In contrast, β-actin DNA amplification exhibited little change within the first 6 min of treatment, but decreased slightly after 12 min (data not shown). The heat-denaturation effect on real-time PCR amplification was also observed in plasmid DNA and was accounted for by the accumulation of single-stranded molecules caused by strand breaks and denaturation (Figure 4B and C). Several practical implications could be derived from the effect of heat-denaturation on mtDNA quantification. For example, the initial heat-denaturing time in PCR amplification could have major impact on real-time PCR analysis of mtDNA content and structural changes. As demonstrated in both plasmid and mtDNA analyses earlier, real-time PCR was more efficient when acting upon relaxed from than upon supercoiled form DNA. Short initial denaturation time would better preserve the original ratios of supercoiled versus relaxed mtDNA, therefore enhancing the sensitivity for structural analysis. However, prolonged initial denaturation could convert all supercoiled DNA into its open-relaxed forms, eliminating structural effects all together. Incorporation of these two strategies could provide a simple new approach to analyze both relaxed form and total mtDNA content simultaneously using real-time PCR (see later).Figure 4.

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