SciCombinator

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Concept: DNA polymerase

186

High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP) on a known template, allowing DNA sequences to encode information about the local cation concentration. In this work we quantify the cation sensitivity of DNAP misincorporation rates, making possible the indirect readout of cation concentration by DNA sequencing. Using multiplexed deep sequencing, we quantify the misincorporation properties of two DNA polymerases - Dpo4 and Klenow exo(-) - obtaining the probability and base selectivity of misincorporation at all positions within the template. We find that Dpo4 acts as a DNA recording device for Mn(2+) with a misincorporation rate gain of ∼2%/mM. This modulation of misincorporation rate is selective to the template base: the probability of misincorporation on template T by Dpo4 increases >50-fold over the range tested, while the other template bases are affected less strongly. Furthermore, cation concentrations act as scaling factors for misincorporation: on a given template base, Mn(2+) and Mg(2+) change the overall misincorporation rate but do not alter the relative frequencies of incoming misincorporated nucleotides. Characterization of the ion dependence of DNAP misincorporation serves as the first step towards repurposing it as a molecular recording device.

Concepts: DNA, Polymerase chain reaction, Chemistry, DNA replication, DNA polymerase, DNA polymerase I, Polymerase, DNA polymerase III holoenzyme

174

In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

Concepts: DNA, Gene expression, Polymerase chain reaction, Molecular biology, DNA replication, DNA polymerase, Genetically modified organism, Genetically modified food

170

Abstract. Visceral leishmaniasis was first reported in Bhutan in 2006. We conducted studies of the parasite, possible vectors and reservoirs, and leishmanin skin test and risk factor surveys in three villages. Nineteen cases were reported from seven districts. Parasite typing yielded two novel microsatellite sequences, both related to Indian L. donovani. In one case village, 40 (18.5%) of 216 participants had positive leishmanin skin test results, compared with 3 (4.2%) of 72 in the other case village and 0 of 108 in the control village. Positive results were strongly associated with the village and increasing age. None of the tested dogs were infected. Eighteen sand flies were collected, 13 Phlebotomus species and 5 Sergentomyia species; polymerase chain reaction for leishmanial DNA was negative. This assessment suggests that endemic visceral leishmaniasis transmission has occurred in diverse locations in Bhutan. Surveillance, case investigations, and further parasite, vector, and reservoir studies are needed. The potential protective impact of bed nets should be evaluated.

Concepts: DNA, Polymerase chain reaction, DNA polymerase, Leishmaniasis, Leishmania, Thermus aquaticus, Phlebotominae, Canine leishmaniasis

169

Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0)) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

Concepts: DNA, Genetics, Cancer, Mutation, Evolution, Mitochondrial DNA, DNA repair, DNA polymerase

168

BACKGROUND: The aim of this study was to optimize quantitative (real-time) polymerase chain reaction (qPCR) assays for 8 major periodontal pathogens, i.e. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micros, Porphyromonas gingivalis, Prevotella intermedia, Tanerella forsythia and Treponema denticola, and of the caries pathogen Streptococcus mutans. RESULTS: Eighteen different primer pairs were analyzed in silico regarding specificity (using BLAST analysis) and the presence of secondary structures at primer binding sites (using mFOLD). The most specific and efficiently binding primer pairs, according to these analyses, were selected for qPCR-analysis to determine amplification efficiency, limit of quantification and intra-run reproducibility. For the selected primer pairs, one for each species, the specificity was confirmed by assessing amplification of DNA extracts from isolates of closely related species. For these primer pairs, the intercycler portability was evaluated on 3 different thermal cyclers (the Applied Biosystems 7300, the Bio-Rad iQ5 and the Roche Light Cycler 480). For all assays on the different cyclers, a good correlation of the standard series was obtained (i.e. r2 >= 0.98), but quantification limits varied among cyclers. The overall best quantification limit was obtained by using a 2 mul sample in a final volume of 10 mul on the Light Cycler 480. CONCLUSIONS: In conclusion, the proposed assays allow to quantify the bacterial loads of S. mutans, 6 periodontal pathogenic species and the genus Fusobacterium.This can be of use in assessing periodontal risk, determination of the optimal periodontal therapy and evaluation of this treatment.

Concepts: DNA, Bacteria, Polymerase chain reaction, Microbiology, Pathogen, DNA replication, DNA polymerase, Oral microbiology

167

Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.

Concepts: DNA, Polymerase chain reaction, Molecular biology, Real-time polymerase chain reaction, DNA replication, DNA polymerase, Laboratory techniques, Primer

152

Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease.

Concepts: DNA, Infectious disease, Polymerase chain reaction, Enzyme, Fungus, DNA polymerase, Thermus aquaticus, Chain reaction

146

In humans, horses, and rodents, an association between pulmonary fibrotic disorders and gammaherpesvirus infection has been suggested. In dogs, canine idiopathic pulmonary fibrosis (CIPF), a progressive fibrotic lung disease of unknown origin and poorly understood pathophysiology, has been reported to occur in West Highland white terriers (WHWTs). The present study investigated the potential association between CIPF and herpesvirus infection. A PCR assay, using a mixture of degenerate and deoxyinosine-substituted primers targeting highly conserved regions of the DNA polymerase gene (DPOL) of herpesviruses, was applied on both lung and blood samples from WHWTs affected with CIPF and controls. Herpesvirus DPOL sequence could not be amplified from any of 46 lung samples (28 affected WHWTs and 18 control dogs of various breeds) and 38 blood samples (19 CIPF WHWTs and 19 control age-matched WHWTs) included. An association between CIPF and herpesvirus infection is therefore unlikely. Investigation of other causes of the disease is warranted.

Concepts: DNA, Polymerase chain reaction, Virus, Fibrosis, DNA polymerase, Idiopathic pulmonary fibrosis, Pulmonary fibrosis, West Highland White Terrier

144

Orf virus infection has been prevalent continuously in the population of wild Japanese serows (Capricornis crispus), goat-like grazing cloven-hoofed mammal species that live mainly in mountainous areas of Japan. Currently, definitive diagnosis of infection requires time-consuming laboratory work. To diagnose rapidly on-site, we developed a field-friendly procedure for the detection of orf virus from oral cavity lesions. DNA was extracted from goat saliva spiked with orf virus as a proxy for Japanese serows by a commercial kit without the use of electricity, and the quality of the extracted DNA was evaluated by conventional polymerase chain reaction (PCR). Extracted DNA was amenable to DNA amplification, the same as when extracted in a laboratory. Next, to find optimal conditions for DNA amplification by loop-mediated isothermal amplification (LAMP), Bst and Csa DNA polymerases and 3 colorimetric indicators for visual diagnosis, hydroxy naphthol blue (HNB), malachite green and D-QUICK, were compared using a portable cordless incubator. The combination of Bst or Csa DNA polymerase with HNB was found to be easiest for visual diagnosis by the naked eye, and viral DNA was successfully amplified from all orf virus strains used. These results suggest that the procedure established here can work completely on-site and can be useful for definitive diagnosis and differentiation of orf virus infection in Japanese serows in remote mountainous areas.

Concepts: DNA, Polymerase chain reaction, Enzyme, DNA replication, Reverse transcriptase, DNA polymerase, Polymerase, Primer

30

Most molecular diagnostic assays require upfront sample preparation steps to isolate the target’s nucleic acids, followed by its amplification and detection using various nucleic acid amplification techniques. Because molecular diagnostic methods are generally rather difficult to perform manually without highly trained users, automated and integrated systems are highly desirable but too costly for use at point-of-care or low-resource settings. Here, we showcase the development of a low-cost and rapid nucleic acid isolation and amplification platform by modifying entry-level 3D printers that cost between $400 and $750. Our modifications consisted of replacing the extruder with a tip-comb attachment that houses magnets to conduct magnetic particle-based nucleic acid extraction. We then programmed the 3D printer to conduct motions that can perform high-quality extraction protocols. Up to 12 samples can be processed simultaneously in under 13 minutes and the efficiency of nucleic acid isolation matches well against gold-standard spin-column-based extraction technology. Additionally, we used the 3D printer’s heated bed to supply heat to perform water bath-based polymerase chain reactions (PCRs). Using another attachment to hold PCR tubes, the 3D printer was programmed to automate the process of shuttling PCR tubes between water baths. By eliminating the temperature ramping needed in most commercial thermal cyclers, the run time of a 35-cycle PCR protocol was shortened by 33%. This article demonstrates that for applications in resource-limited settings, expensive nucleic acid extraction devices and thermal cyclers that are used in many central laboratories can be potentially replaced by a device modified from inexpensive entry-level 3D printers.

Concepts: DNA, Polymerase chain reaction, Molecular biology, Nucleic acid, DNA polymerase, Thermus aquaticus, Taq polymerase, Thermal cycler