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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

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).
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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).

Mentions: pBR322 plasmid DNA was digested with enzymes that relax the supercoiled form (Figure 1A). EcoR I (New England Biolabs, Ipswich, MA) that has a single restriction site in pBR322 DNA was used to generate its linear forms. N.BstNB1 (New England Biolabs) that has two nicking sites in pBR322 DNA was used to generate the nicked circular form. Topoisomerase I (Invitrogen) relaxes negative supercoiling and was used to generate relaxed closed-circular topoisomers of plasmid DNA. Total genomic DNA from LNCaP was also digested with EcoR1 to linearize mtDNA. Digested plasmid and total genomic DNA was recovered by ethanol precipitation and quantified using the PicoGreen dsDNA Quantification Kit.Figure 1.


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)

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

License
Show All Figures
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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).
Mentions: pBR322 plasmid DNA was digested with enzymes that relax the supercoiled form (Figure 1A). EcoR I (New England Biolabs, Ipswich, MA) that has a single restriction site in pBR322 DNA was used to generate its linear forms. N.BstNB1 (New England Biolabs) that has two nicking sites in pBR322 DNA was used to generate the nicked circular form. Topoisomerase I (Invitrogen) relaxes negative supercoiling and was used to generate relaxed closed-circular topoisomers of plasmid DNA. Total genomic DNA from LNCaP was also digested with EcoR1 to linearize mtDNA. Digested plasmid and total genomic DNA was recovered by ethanol precipitation and quantified using the PicoGreen dsDNA Quantification Kit.Figure 1.

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