Effect of DNA polymerases on PCR-DGGE patterns

Balázs, Margit; Rónavári, Andrea; Németh, Alexandra; Bihari, Zoltán; Rutkai, Edit; Bartos, Péter; Kiss, István; Szvetnik, Attila

doi:10.1016/j.ibiod.2012.05.011

Cited by 21 publications

(12 citation statements)

References 45 publications

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“…In microbial ecology, culture dependent methods have characterised soil fungal communities but these detect only a fraction (ca 1%) of the total populations [22,23].…”

mentioning

confidence: 99%

Soil fungal community shift evaluation as a potential cadaver decomposition indicator

Chimutsa

Olakanye

Thompson

et al. 2015

Forensic Science International

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“…In microbial ecology, culture dependent methods have characterised soil fungal communities but these detect only a fraction (ca 1%) of the total populations [22,23].…”

mentioning

confidence: 99%

Soil fungal community shift evaluation as a potential cadaver decomposition indicator

Chimutsa

Olakanye

Thompson

et al. 2015

Forensic Science International

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“…; Balazs et al . ). It is therefore crucial to establish whether sample processing prior to DGGE analysis suits the application and produces PCR products of the desired quality and quantity.…”

Section: Resultsmentioning

confidence: 97%

“…There are many pitfalls when analysing microbial diversity using PCR-based analyses (Wintzingerode et al 1997;Pontes et al 2007;Harwood et al 2014). Molecular biology is also a fast evolving discipline and many new hurdles in DGGE applications have recently come to light (Ascher et al 2010;Balazs et al 2013). It is therefore crucial to establish whether sample processing prior to DGGE analysis suits the application and produces PCR products of the desired quality and quantity.…”

Section: Resultsmentioning

confidence: 99%

The use of PCR-DGGE to determine bacterial fingerprints for poultry and red meat abattoir effluent

Smidt

2016

Letters in Applied Microbiology

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This study was the first to demonstrate the application of denaturing gradient gel electrophoresis (DGGE) to construct bacterial diversity fingerprints for high-throughput abattoir effluents. Proved redundancy of fat removal as PCR inhibitor and change in diversity similarity introduced by nested PCR approach. The importance of limiting excessive handling/processing which could lead to misrepresented diversity profiles was emphasized.

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“…Yet, biased profiles can be highly reproducible depending on the polymerase used for the reaction [111]. Although unsupported by experimental data, criteria such as the maximum yield of a PCR product [116] or the highest variety of amplicon types [117], [118] are often used for multi-template PCR optimization. Representation of a sequence by more than one clone, preferably obtained in independent PCRs, is used as a threshold to assign new alleles of the gene coding for the major histocompatibility complex (MHC) in vertebrates [119].…”

Section: Factors That Can Increase Artifacts Formation In Multi-templmentioning

confidence: 99%

Multi-template polymerase chain reaction

Kalle

Kubista

Rensing

2014

Biomolecular Detection and Quantification

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PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

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Effect of DNA polymerases on PCR-DGGE patterns

Cited by 21 publications

References 45 publications

Soil fungal community shift evaluation as a potential cadaver decomposition indicator

Soil fungal community shift evaluation as a potential cadaver decomposition indicator

The use of PCR-DGGE to determine bacterial fingerprints for poultry and red meat abattoir effluent

Multi-template polymerase chain reaction

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