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

Chen, Jinsong; Kadlubar, Fred F.; Chen, Junjian Z.

doi:10.1093/nar/gkm010

Cited by 84 publications

(92 citation statements)

References 54 publications

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“…We suspected that as the RCA product grew too long and formed a very compact random coil, some of the digestion sites might be embedded too deep inside the coil and become inaccessible to the digestion enzyme. The phenomenon that the supercoiled DNA was a poor substrate for primer and enzyme binding was also observed in using real-time PCR for mitochondrial DNA quantification [23]. The digested product migrated later than the 100 nt peak from the ssDNA ladder in our case, even though it should have a similar size as the padlock probe (73 nt).…”

Section: Reaction Optimizationsupporting

confidence: 58%

CE combined with rolling circle amplification for sensitive DNA detection

Zhong

2008

Electrophoresis

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Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.

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Section: Reaction Optimizationsupporting

confidence: 58%

CE combined with rolling circle amplification for sensitive DNA detection

Zhong

2008

Electrophoresis

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“…The real-time PCR signal from supercoiled DNA is less than that for linear conformations (32); therefore, in contrast to published work by Navarro et al (25), we used a linear plasmid for the preparation of a standard curve. The detection limit of the bcsp31 real-time PCR in this work was 10 fg, which noticeably improved on results reported by Debeaumont et al (11).…”

Section: Discussionmentioning

confidence: 99%

Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

et al. 2014

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cRapid and effective diagnosis of brucellosis is a challenge for clinicians. Even when diagnosis is on time and therapy is initiated, meticulous follow-up appointments are crucial for ensuring the efficacy of the treatment. Due to shortcomings of serological methods, molecular diagnosis, especially real-time PCR, is becoming a main approach in laboratory diagnostics. Thus, the development of efficient procedures and standardization of the PCR tests will have a great impact on the precise detection and quantification of bacterial DNA loads, which is valuable for the medical management of brucellosis patients. We developed a new TaqMan real-time PCR directed to bcsp31, a shared gene of the brucellae. The bcsp31 gene fragment was cloned into pJET1.2. Recombinant pJET1.2-bcsp31 was linearized by HindIII digestion, and the product was used for the preparation of a standard curve. A panel of Brucella spp. and non-Brucella pathogens was tested. No bacterial genomes other than those of the brucellae were detected. According to the results, specificity of the method was 100%. In a clinical assessment, the positive-control group comprised 37 patients with microbiologically confirmed brucellosis, and 25 healthy individuals served as the negative-control group. By the end of the treatment period, there was a significant decrease in the DNA load of the 37 brucellosis patients, which persisted for the 4 weeks of monitoring after treatment, suggesting that our proposed method is an efficient monitoring tool. Serum samples prior to any treatment were collected from the 25 serologically suspicious patients and assessed by our method; 72% of these patients tested positive for brucellosis.T he brucellae are facultative, intracellular, Gram-negative bacteria that infect animals and humans (1). Although it has been eradicated in some developed countries, brucellosis still remains a major problem worldwide. Diagnosis of the disease presents a major challenge to clinical and veterinary services (2, 3). Brucella spp. are able to survive within host cells, leading to relapses and focal complications (1). Early diagnosis of brucellosis results in early initiation of therapy, which plays a key role in the success of the control and eradication programs; nonetheless, accurate diagnosis requires reliable and sensitive diagnostic tools (4). The intracellular localization of the bacteria and the slow evolution of the disease hamper the usefulness of blood cultures (5). Despite being the gold standard, isolation of brucellae from blood culture or other sterile body fluids has a sensitivity of about 70% (4, 6). Serodiagnostic assays are principally based on antibodies against the Brucella lipopolysaccharide (LPS) and have high sensitivity but low specificity. The low specificity may be due to the crossreaction of antibodies with LPS from other bacterial species and subsensitive or high-immunity reactions, depending on the subclinical or endemic prevalence of the disease (4, 6, 7). The presence of IgG and IgM for months after therapy complicates...

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“…After 2 minutes of heat denaturation at 94°C, intact mtDNA remains largely in a supercoiled state (being resistant to amplification by PCR), while damaged mtDNA is relaxed and can be amplified. After 6 minutes at 94°C, all mtDNA is relaxed and can be amplified (Chen et al, 2007). Thus, relative mtDNA damage was measured as the ratio of amplification efficiencies for the 83 bp amplicon [primer nucleotide sequences 5′-GATTTGGGTACCACCCAAGTATTG-3′ (16042-16064) and 5′-AATATTCAT -GGTGGCTGGCATGTA-3′ (16125-16102)] following 2 and 6 minutes of heat denaturation.…”

Section: Mtdna Damage and Copy Numbermentioning

confidence: 99%

Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress

Ahmed

Passos

Birket

et al. 2008

Journal of Cell Science

424

431

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Telomerase is a ribonucleoprotein that counteracts telomere shortening and can immortalise human cells. There is also evidence for a telomere-independent survival function of telomerase. However, its mechanism is not understood. We show here that TERT, the catalytic subunit of human telomerase, protects human fibroblasts against oxidative stress. While TERT maintains telomere length under standard conditions, telomeres under increased stress shorten as fast as in cells without active telomerase. This is because TERT is reversibly excluded from the nucleus under stress in a dose- and time-dependent manner. Extranuclear telomerase colocalises with mitochondria. In TERT-overexpressing cells, mtDNA is protected, mitochondrial membrane potential is increased and mitochondrial superoxide production and cell peroxide levels are decreased, all indicating improved mitochondrial function and diminished retrograde response. We propose protection of mitochondria under mild stress as a novel function of TERT.

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

Cited by 84 publications

References 54 publications

CE combined with rolling circle amplification for sensitive DNA detection

CE combined with rolling circle amplification for sensitive DNA detection

Efficient Diagnosis and Treatment Follow-Up of Human Brucellosis by a Novel Quantitative TaqMan Real-Time PCR Assay: a Human Clinical Survey

Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress

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