Effects of ethidium bromide and SYBR® Green I on different polymerase chain reaction systems

Nath, Kamalendu; Sarosy, Joseph W.; Hahn, James K.; Como, Charles J. Di

doi:10.1016/s0165-022x(99)00033-0

Cited by 73 publications

(42 citation statements)

References 24 publications

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“…The SYBR Green I dye is inhibitory to Taq enzyme, which may impose certain limitations on the fluorescent signal and sensitivity of detection. 38,39 We found earlier that the Taq mutants OT and OKT were able to tolerate extremely high concentrations of SYBR Green in crude samples, especially blood. 27 This finding was very helpful, because using an excess of the dye could compensate for the quenching effect of the blood components.…”

Section: Discussionmentioning

confidence: 98%

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Zhang

Kermekchiev

Barnes

2010

The Journal of Molecular Diagnostics

124

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PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition , enhance PCR amplification , and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent , L-carnitine , D-(؉)-trehalose , and heparin. These cocktails , in combination with two inhibitor-resistant Taq mutants , OmniTaq and Omni Klentaq , enabled efficient amplification of exogenous , endogenous , and high-GC content DNA targets directly from crude samples containing human plasma , serum , and whole blood without DNA purification. In the presence of these enhancer cocktails , the mutant enzymes were able to tolerate at least 25% plasma , serum , or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples , both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial

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Section: Discussionmentioning

confidence: 98%

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Zhang

Kermekchiev

Barnes

2010

The Journal of Molecular Diagnostics

124

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“…Initially, there was concern that contamination of the PCRs with curing agent may cause this effect, as both EtBr and AcOr have been shown to inhibit PCR by intercalation (Nath et al, 2000). As plasmid topology limits such intercalation, it was postulated that preferential inhibition of the omcB assay (during the first PCR cycle) may occur.…”

Section: Discussionmentioning

confidence: 99%

The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents

et al. 2005

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A 7?5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1-L3), ten trachoma isolates (serovars A-C) and nine urogenital isolates (serovars D-J). The results indicated an average plasmid copy number of 4?0±0?8 (mean±95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7?6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7?4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1?3±0?2 plasmids per chromosome (mean±95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number. INTRODUCTIONMembers of the Chlamydiaceae are obligately intracellular, Gram-negative bacteria of significant importance in both human and animal pathogenesis. These organisms exhibit a unique developmental cycle, in which a metabolically active reticulate body (RB) gives rise to a dense, infectious elementary body (EB) (Rockey & Matsumoto, 2000). The family comprises two genera, Chlamydophila and Chlamydia (Everett et al., 1999), although this classification remains controversial (Schachter et al., 2001).Chlamydophila pneumoniae is an important human pathogen implicated in arterial disease (Saikku et al., 1992); infection of the respiratory tract causes an atypical pneumonia (Hahn et al., 2002). Although predominantly a human pathogen, C. pneumoniae strains have been isolated from animal species, e.g. equines (Everett et al., 1999;Storey et al., 1993; Wills et al., 1990) and koala (Wardrop et al., 1999).Chlamydia trachomatis is not only the major infectious agent of preventable blindness in the developing world (Thylefors et al., 1995), but also the commonest cause of non-specific urethritis in developed countries (Burstein & Zenilman, 1999). Sexually transmitted disease caused by C. trachomatis is frequently undiagnosed in women, whereupon an ascending infection may lead to pelvic inflammatory disease, salpingitis and consequent infe...

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“…Between the wide variety in commerce, most of the Real-time PCR approaches are based on the use of SYBR Ò Green I as DNA dye. SYBR Ò Green I is a high specific dye but its use has been shown to have some disadvantages, such as the inhibition of PCR reactions at high concentration [10,11], negative effect on DNA melting temperature [10,12], and preferential binding to GC-rich DNA sequences [12]. An alternative DNA dye is the EvaGreen Ò , which has been demonstrated having a higher reaction efficiency at different concentration as well as better melting curves with sharper peaks compared with the SYBR Green chemistry [13].…”

Section: Introductionmentioning

confidence: 99%

EvaGreen Real-time PCR protocol for specific ‘Candidatus Phytoplasma mali’ detection and quantification in insects

Monti

Martini

Tedeschi

2013

Molecular and Cellular Probes

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Effects of ethidium bromide and SYBR® Green I on different polymerase chain reaction systems

Cited by 73 publications

References 24 publications

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq

The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents

EvaGreen Real-time PCR protocol for specific ‘Candidatus Phytoplasma mali’ detection and quantification in insects

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