Polymerase chain reaction.
TL;DR: It is important to understand the basic principles of PCR and how its use can be modified to provide for sophisticated analysis of genes and the genome.
About: This article is published in Journal of Investigative Dermatology.The article was published on 2013-03-01 and is currently open access. It has received 538 citations till now. The article focuses on the topics: Applications of PCR & In silico PCR.
Citations
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TL;DR: This review presents an opportunity to fill a knowledge gap for an extremely important research area; discussing the main techniques, biology, chemistry, miniaturisation, sensing and the emerging state-of-the-art research and developments for detection of pathogens in food, water, blood and faecal samples.
Abstract: The testing and rapid detection of pathogenic organisms is a crucial protocol in the prevention and identification of crises related to health, safety and wellbeing. Pathogen detection has become one of the most challenging aspects in the food and water industries, because of the rapid spread of waterborne and foodborne diseases in the community and at significant costs. With the prospect of inevitable population growth, and an influx of tourism to certain water bodies testing will become a requirement to control and prevent possible outbreaks of potentially fatal illnesses. The legislation is already particularly rigorous in the food industry, where failure to detect pathogenic materials represents a catastrophic event, particularly for the elderly, very young or immune-compromised population types. In spite of the need and requirement for rapid analytical testing, conventional and standard bacterial detection assays may take up to seven days to yield a result. Given the advent of new technologies, biosensors, chemical knowledge and miniaturisation of instrumentation this timescale is not acceptable. This review presents an opportunity to fill a knowledge gap for an extremely important research area; discussing the main techniques, biology, chemistry, miniaturisation, sensing and the emerging state-of-the-art research and developments for detection of pathogens in food, water, blood and faecal samples.
256 citations
TL;DR: The potential impact of SERS in addressing unmet needs in pathogen diagnostics both in a traditional clinical setting and in the point of care (POC) arena is examined.
Abstract: Surface-enhanced Raman scattering (SERS) has enabled the detection of pathogens and disease markers at extremely low levels. This review examines the potential impact of SERS in addressing unmet needs in pathogen diagnostics both in a traditional clinical setting and in the point of care (POC) arena. It begins by describing the strengths and weaknesses of today's diagnostics technologies in order to set a contextual stage for an overview which highlights a few of the many recent developments using SERS in biodefense, human and animal health, and monitoring food and water safety. These sections are followed by discussions of the challenges for the translation of these developments to POC settings, including the performance attributes and metrics for quantification of analytical and clinical figures of merit (e.g., limit of detection and clinical accuracy), and the pathways for large-scale test validation and the build-out of instrumentation and tests kits for POC deployment.
191 citations
TL;DR: The portability of biosensors for on-site diagnosis is limited due to various issues, including sample preparation techniques, fluid-handling techniques, the limited lifetime of biological reagents, device packaging, integrating electronics for data collection/analysis, and the requirement of external accessories and power.
Abstract: The significance of microfluidics-based and microelectromechanical systems-based biosensors has been widely acknowledged, and many reviews have explored their potential applications in clinical diagnostics, personalized medicine, global health, drug discovery, food safety, and forensics. Because health care costs are increasing, there is an increasing need to remotely monitor the health condition of patients by point-of-care-testing. The demand for biosensors for detection of biological warfare agents has increased, and research is focused on ways of producing small portable devices that would allow fast, accurate, and on-site detection. In the past decade, the demand for rapid and accurate on-site detection of plant disease diagnosis has increased due to emerging pathogens with resistance to pesticides, increased human mobility, and regulations limiting the application of toxic chemicals to prevent spread of diseases. The portability of biosensors for on-site diagnosis is limited due to various issues, including sample preparation techniques, fluid-handling techniques, the limited lifetime of biological reagents, device packaging, integrating electronics for data collection/analysis, and the requirement of external accessories and power. Many microfluidic, electronic, and biological design strategies, such as handling liquids in biosensors without pumps/valves, the application of droplet-based microfluidics, paper-based microfluidic devices, and wireless networking capabilities for data transmission, are being explored.
162 citations
TL;DR: The finding reveals that the uterine microbiota in dairy cows varies according with health status and DPP, and adds further support to the hypothesis that there is uterine contamination with diverse bacterial groups following calving and emphasizes the role of unidentified microorganisms in this context.
Abstract: The diversity of the uterine bacterial composition in dairy cows is still poorly understood, although the emerging picture has shown to be increasingly complex. Understanding the complexity and ecology of microorganisms in the uterus of postpartum dairy cows is critical for developing strategies to block their action in reproductive disorders, such as metritis/endometritis. Here, we used PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and DNA pyrosequencing to provide a comprehensive description of the uterine bacterial diversity and compare its succession in healthy, metritic and endometritic Holstein dairy cows at three intervals following calving. Samples were collected from 16 dairy cows housed in a dairy farm located in upstate New York. PCR-DGGE revealed a complex profile with extensive differences in the community structure. With few exceptions, clustering analysis grouped samples from cows presenting the same health status. Analysis of >65,000 high-quality 16S rRNA gene sequences showed that the uterine bacterial consortia, regardless of the health status, is mainly composed of members of the phyla Bacteroidetes, Fusobacteria, Firmicutes, Proteobacteria, and Tenericutes. In addition to these co-dominant phyla, sequences from Spirochaetes, Synergistetes, and Actinobacteria appear less frequently. It is possible that some sequences detected in the uterine fluid resulted from the presence of fecal or vaginal contaminants. Overall, the bacterial core community was different in uterine fluid of healthy cows, when compared to cows suffering from postpartum diseases, and the phylogenetic diversity in all the combined samples changed gradually over time. Particularly at the 34–36 days postpartum (DPP), the core community seemed to be specific for each health status. Our finding reveals that the uterine microbiota in dairy cows varies according with health status and DPP. Also, it adds further support to the hypothesis that there is uterine contamination with diverse bacterial groups following calving and emphasizes the role of unidentified microorganisms in this context.
131 citations
Patent•
12 Apr 2012TL;DR: Safe-SeqS as mentioned in this paper assigns a unique identifier (UID) to each template molecule and amplifies each uniquely tagged template molecule to create UID-families, which increases the sensitivity of massively parallel sequencing instruments for this purpose.
Abstract: The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≧95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.
120 citations
References
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TL;DR: This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation and the conceptual framework for data analysis methods is presented to demystify these analysis techniques.
Abstract: Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput.
1,149 citations
"Polymerase chain reaction." refers background or methods in this paper
...It indicates how much of a specific DNA or gene is present in the sample. qPCR allows for both detection and quantification of the PCR product in real time, while it is being synthesized (VanGuilder et al., 2008)....
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...qPCR allows for both detection and quantification of the PCR product in real time, while it is being synthesized (VanGuilder et al., 2008)....
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TL;DR: I stumbled across a process that could make unlimited numbers of copies of genes, a process now known as the polymerase chain reaction (PCR), one Friday night in April, 1983, as I gripped the steering wheel of my car and snaked along a moonlit mountain road into northern California's redwood country.
Abstract: S ometimes a good idea comes to you when you are not looking for it. Through an improbable combination of coincidences, naivete and lucky mistakes, such a revelation came to me one Friday night in April, 1983, as I gripped the steering wheel of my car and snaked along a moonlit mountain road into northern California's redwood country. That was how I stumbled across a process that could make unlimited numbers of copies of genes, a process now known as the polymerase chain reaction (PCR). Beginningwith a single molecule of the genetic material DNA,the PCRcan generate 100 billion similar molecules in an afternoon. The reaction is easy to execute: it requires no more than a test tube, a few simple reagents and a source of heat. The DNAsample that one wishes to copy can be pure, or it can be a minute part of an extremely complex mixture of biological materials. The DNAmay come from a hospi-
1,006 citations
"Polymerase chain reaction." refers background in this paper
...The advent of the polymerase chain reaction (PCR) radically transformed biological science from the time it was discovered (Mullis, 1990)....
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...Lilit Garibyan1 and Nidhi Avashia2 Journal of Investigative Dermatology (2013) 133, e6. doi:10.1038/jid.2013.1 The advent of the polymerase chain reaction (PCR) radically transformed biological science from the time it was discovered (Mullis, 1990)....
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...Kary Mullis, who conceptualized the PCR assay, explained that it “lets you pick the piece of DNA you’re interested in and have as much of it as you want” (Mullis, 1990)....
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TL;DR: Future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology are offered, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.
724 citations
"Polymerase chain reaction." refers background in this paper
...For example, in a study to link distinct taxa within the microbial community to specific metabolic processes, stable isotope probing was combined with qPCR (Postollec et al., 2011; Smith and Osborn, 2009)....
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...Because it is highly sensitive, any form of contamination of the sample by even trace amounts of DNA can produce misleading results (Vogel et al., 2012; Smith and Osborn, 2009)....
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TL;DR: The basic concepts, chemistries, and instrumentation of real-time PCR are discussed and the present applications and future perspectives for this technology in biomedical sciences and in life science education are included.
Abstract: In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of ...
605 citations
"Polymerase chain reaction." refers methods in this paper
...Real-time PCR can be combined with reverse transcription, which allows messenger RNA to be converted into cDNA (i.e., reverse transcription), after which quantification of the cDNA is performed with qPCR (Valasek and Repa, 2005)....
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..., reverse transcription), after which quantification of the cDNA is performed with qPCR (Valasek and Repa, 2005)....
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TL;DR: Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression.
378 citations
"Polymerase chain reaction." refers background in this paper
...For example, in a study to link distinct taxa within the microbial community to specific metabolic processes, stable isotope probing was combined with qPCR (Postollec et al., 2011; Smith and Osborn, 2009)....
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