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Methods in Molecular Biology (Clifton,... 2022Amplification of different nucleic acid targets in the same reaction (multiplex polymerase chain reaction) is challenging but an extremely useful tool especially for...
Amplification of different nucleic acid targets in the same reaction (multiplex polymerase chain reaction) is challenging but an extremely useful tool especially for viroid diagnosis. In the amplification mixtures, several pairs of primers work together in the same conditions to detect different targets. Here, we describe the development and use of a multiplex reverse transcription polymerase chain reaction protocol highlighting the most crucial factors that can significantly affect the quality of the method. First, particular attention must be paid to primer design. Then, the amplification mixture and temperature conditions must be calibrated precisely to avoid cross reactivity or loss in sensitivity. Finally, the detection system of the amplification results must allow a specific identification of the amplified target(s).
Topics: Multiplex Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Viroids
PubMed: 34845694
DOI: 10.1007/978-1-0716-1464-8_15 -
Biochimie Jun 2022Polymerase chain reaction is an important tool in molecular biology. Although the principles of the technique are relatively simple, amplifying complex or long DNA... (Review)
Review
Polymerase chain reaction is an important tool in molecular biology. Although the principles of the technique are relatively simple, amplifying complex or long DNA segments can be challenging. A variety of PCR additives are used to improve the performance and yield of difficult PCRs. Each PCR additive has unique properties and enhances PCR through a different mode of action. They are used to either improve PCR sensitivity, efficiency, and specificity, or mitigate the effects of PCR inhibitors. In this review, we categorise known PCR additives into four main groups. The first three groups comprise PCR additives with well-defined mechanisms, namely those that facilitate the amplification of GC-rich sequences, counteract the detrimental effects of PCR inhibitors, or alter PCR kinetics (nanomaterials). The fourth group is a loose mix of additives with unclear mechanisms of action. Then, we discuss how these additives may be used to tackle specific PCR-related challenges, particularly those associated with long-range PCR. We conclude the review with added insights into the use of PCR additives in enhancing the synthesis of complex and long DNA fragments.
Topics: DNA; GC Rich Sequence; Polymerase Chain Reaction; Regulatory Sequences, Nucleic Acid
PubMed: 35231536
DOI: 10.1016/j.biochi.2022.02.009 -
Trends in Biotechnology Jul 2019Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the... (Review)
Review
Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.
Topics: Costs and Cost Analysis; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Time
PubMed: 30654913
DOI: 10.1016/j.tibtech.2018.12.002 -
Biosensors May 2021Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR)... (Review)
Review
Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.
Topics: Animals; COVID-19; COVID-19 Nucleic Acid Testing; CRISPR-Cas Systems; Equipment Design; Humans; Point-of-Care Testing; Polymerase Chain Reaction; SARS-CoV-2
PubMed: 34062874
DOI: 10.3390/bios11050141 -
Mini Reviews in Medicinal Chemistry 2023Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the... (Review)
Review
Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.
Topics: Humans; Molecular Biology; Water; Freeze Drying; Polymerase Chain Reaction; Cryopreservation; Point-of-Care Systems
PubMed: 35927908
DOI: 10.2174/1389557522666220802144057 -
Critical Reviews in Biotechnology May 2023Digital polymerase chain reaction (dPCR) technology has provided a new technique for molecular diagnostics, with superior advantages, such as higher sensitivity,... (Review)
Review
Digital polymerase chain reaction (dPCR) technology has provided a new technique for molecular diagnostics, with superior advantages, such as higher sensitivity, precision, and specificity over quantitative real-time PCRs (qPCR). Eight companies have offered commercial dPCR instruments: Fluidigm Corporation, Bio-Rad, RainDance Technologies, Life Technologies, Qiagen, JN MedSys Clarity, Optolane, and Stilla Technologies Naica. This paper discusses the working principle of each offered dPCR device and compares the associated: technical aspects, usability, costs, and current applications of each dPCR device. Lastly, up-and-coming dPCR technologies are also presented, as anticipation of how the dPCR device landscape may likely morph in the next few years.
Topics: Real-Time Polymerase Chain Reaction; Biotechnology; Commerce
PubMed: 35291902
DOI: 10.1080/07388551.2022.2037503 -
Bioanalysis Jun 2022Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the...
Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.
Topics: Biological Assay; Real-Time Polymerase Chain Reaction
PubMed: 35703321
DOI: 10.4155/bio-2022-0109 -
Clinical Chemistry Aug 2020Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013,...
Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.
Topics: Guidelines as Topic; Humans; Nucleic Acids; Polymerase Chain Reaction; Publications
PubMed: 32746458
DOI: 10.1093/clinchem/hvaa125 -
Biosensors & Bioelectronics Sep 2022Digital PCR (dPCR) is built on partitioning reagent to the extent that single template molecules are amplified and visualized individually, whereby offers higher... (Review)
Review
Digital PCR (dPCR) is built on partitioning reagent to the extent that single template molecules are amplified and visualized individually, whereby offers higher precision and other better indicators than the former PCR techniques. Accordingly, dPCR is particular suited for precision medicine applications that require accurate molecular characterization with high sensitivity. This review aims to summarize different applications of dPCR in precision medicine. The state-of-the-art progress of dPCR technique is first introduced, including novel prototype machines and dPCR-integrated biochips. Then the clinical applications based on dPCR technique are briefly described, for instance, detecting biomarkers from tissues and various biopsies components including cell free DNA, circulating tumor cells, extracellular vesicles, and proteins. These emerging dPCR applications have been accepted as auxiliary diagnostic methods in various areas like oncology, infectious disease, and the like. Meanwhile, a usage overview is provided, focusing on successful clinical pilot studies that dPCR is utilized to improve the performances of rare event detection, fine resolution of gene expression analysis, and multiplexing. Finally, some implications and challenges in future research concerning dPCR technique are also discussed.
Topics: Biosensing Techniques; Polymerase Chain Reaction; Precision Medicine; Technology
PubMed: 35598553
DOI: 10.1016/j.bios.2022.114344 -
BioTechniques Jul 2020Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids...
Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.
Topics: DNA; Emulsions; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Silicon Dioxide
PubMed: 32338528
DOI: 10.2144/btn-2019-0161