A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA

Niederstätter, Harald; Köchl, Silvano; Grubwieser, Petra; Pavlic, Marion; Steinlechner, Martin; Parson, Walther

doi:10.1016/j.fsigen.2006.10.007

Cited by 110 publications

(71 citation statements)

References 9 publications

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“…One negative extraction control was added for each extraction batch. The extraction volume was 200 L. A singleplex real-time PCR assay amplifying a 156-bp gene fragment was used (16,17). The polymerases investigated were Ampli Taq Gold (modified Taq ), Bio-X-Act Short (undisclosed blend of enzymes) (Bioline, London, UK), Ex Taq Hot Start (modified Taq ) (Takara Bio Inc., Shiga, Japan), KAPA2G Robust ( Taq mutant) (KAPA Biosystems, Cape Town, South Africa), Omni Taq ( Taq mutant) (DNA Polymerase Technologies, St. Louis, MO, USA), PicoMaxx High Fidelity (a mixture of recombinant Taq and cloned Pyrococcus furiosus polymerase) (Stratagene, La Jolla, CA, USA), r Tth (recombinant Tth) (Applied Biosystems), Taq , and Tth (both, Roche Diagnostics, Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

Improved Forensic DNA Analysis Through the use of Alternative DNA Polymerases and Statistical Modeling of DNA Profiles

et al. 2009

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DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.

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Section: Methodsmentioning

confidence: 99%

Improved Forensic DNA Analysis Through the use of Alternative DNA Polymerases and Statistical Modeling of DNA Profiles

et al. 2009

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“…[40,183]). Furthermore, recent progress in real-time quantitative PCR technology has enabled sensitive analysis of mtDNA copy number in small sample volumes [195]. By this method mtDNA content is quantified relative to nDNA, yielding a mitochondrial-to-nuclear DNA ratio as measure of mtDNA copy number, which was used as a measure of mitochondrial content in animal and clinical studies [40,[196][197][198].…”

Section: Biochemical Biomarkersmentioning

confidence: 99%

Regulation and Quantification of Cellular Mitochondrial Morphology and Content

Tronstad¹,

Nooteboom²,

Nilsson³

et al. 2014

CPD

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Running title: Regulation and quantification of mitochondrial morphology and content. Word count: 15,454 (total)Characters: 107,091 (including spaces); 92,067 (excluding spaces) Keywords: mitochondrial biogenesis, mitochondrial dynamics, mitochondrial degradation, living cells, TMRM, microscopy, segmentation, image analysis.Page 2 of 37 ABSTRACTMitochondria play a key role in signal transduction, redox homeostasis and cell survival, which extends far beyond their classical functioning in ATP production and energy metabolism. In living cells, mitochondrial content ("mitochondrial mass") depends on the cell-controlled balance between mitochondrial biogenesis and degradation. These processes are intricately linked to changes in net mitochondrial morphology and spatiotemporal positioning ("mitochondrial dynamics"), which are governed by mitochondrial fusion, fission and motility. It is becoming increasingly clear that mitochondrial mass and dynamics, as well as its ultrastructure and volume, are mechanistically linked to mitochondrial function and the cell. This means that proper quantification of mitochondrial morphology and content is of prime importance in understanding mitochondrial and cellular physiology in health and disease. This review first presents how cellular mitochondrial content is regulated at the level of mitochondrial biogenesis, degradation and dynamics. Next we discuss how mitochondrial dynamics and content can be analyzed with a special emphasis on quantitative live-cell microscopy strategies.

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“…Unless otherwise specified, the following reagents were included: 1X Immobuffer (Bioline Reagents Ltd., template DNA. For detection, either an EG dye (Biotium Inc., Hayward, CA, USA) or a 6FAM-probe (RB1, purchased from Life Technologies)[18] was used unless otherwise specified. The same qPCR conditions were used for all tests with a heat-activating step at 95°C for 15 min followed by 55 cycles of 10 s at 95°C, 20 s at 60°C and 30 s at 72°C.…”

mentioning

confidence: 99%

Humic substances cause fluorescence inhibition in real-time polymerase chain reaction

Sidstedt

Jansson

Nilsson

et al. 2015

Analytical Biochemistry

101

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Real-time polymerase chain reaction (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, that is, amplification inhibition. Humic substances (HS) are well-known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, that is, quench the fluorescence signal of double-stranded DNA (dsDNA) binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I, and SYTO 82, generating lowered amplification plots, although amplicon production was unaffected. For EvaGreen, 500 ng of HA quenched nearly all fluorescence, whereas 1000 ng of HA completely inhibited amplification when applying Immolase DNA polymerase with bovine serum albumin (BSA). Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.

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A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA

Cited by 110 publications

References 9 publications

Improved Forensic DNA Analysis Through the use of Alternative DNA Polymerases and Statistical Modeling of DNA Profiles

Improved Forensic DNA Analysis Through the use of Alternative DNA Polymerases and Statistical Modeling of DNA Profiles

Regulation and Quantification of Cellular Mitochondrial Morphology and Content

Humic substances cause fluorescence inhibition in real-time polymerase chain reaction

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