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Digital PCR: theory and applications
... If the dilution is sufficiently high, the number of positive signals will directly reflect the initial template number. However, if the dilution is not sufficiently high, there might be a possibility of the presence of multiple targets in a single partition [56]. A simple addition of all the positive signals will lead to inaccurate quantification in such cases. ...
... Reagents are pipetted into the through-holes and are retained via surface tension. The Results are not affected by any of these factors [21] Advantages Possibility of end-point detection as well as detection during reaction Used as a molecular diagnostic tool for disease diagnosis Multiplexing potential [26] Widely used molecular diagnostic format [34] Ability to monitor the progress of PCR reaction [33] Possibility to precisely measure the amount of amplicon at each cycle Accurate quantification of the amount of starting material [27] Increased dynamic range of detection [26] Single step amplification and detection No post-PCR manipulation required [61] No references or standard curve [35] More precise than real-time PCR [13,42] Improved interlaboratory commutability [18] Highly tolerant to inhibitors [20] Less affected by poor amplification efficiency [56] Better detection of low-copynumber variants [59] Limitations Prone to inhibitors and nontarget NA [27] Poor Precision Low sensitivity [32] Short dynamic range <2 logs Low resolution [34] Not automated Size-based discrimination only, i.e., results are not quantitative Requires post-PCR processing Prone to inhibitors and nontarget NA [32] Expensive detection equipment and consumables [31] Requirement of fluorescent probe [65] Restricted to referral laboratory with good financial support Limited reaction mixture volume [42] High contamination risk More complex to perform compared to qPCR [13] More expensive instrumentation and reagents than qPCR [13] Smaller dynamic range [35] Molecular dropout [13] Lower specimen throughput [37] OpenArray plate is then covered with a lid and analyzed via real-time or end-point measurement on the QuantStudio 12 K Flex system [61]. Several research groups have used Quantstudio for their analysis: the accurate quantification of DNA copy number of a certified pNIM-001 plasmid DNA using QuantStudio 12k dPCR platform was performed by Dong et al. ...
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.
Genetic molecular testing is starting to gain traction as part of standard clinical practice for dogs with cancer due to its multi-faceted benefits, such as potentially being able to provide diagnostic, prognostic and/or therapeutic information. However, the benefits and ultimate success of genomic analysis in the clinical setting are reliant on the robustness of the tools used to generate the results, which continually expand as new technologies are developed. To this end, we review the different materials from which tumour cells, DNA, RNA and the relevant proteins can be isolated and what methods are available for interrogating their molecular profile, including analysis of the genetic alterations (both somatic and germline), transcriptional changes and epigenetic modifications (including DNA methylation/acetylation and microRNAs). We also look to the future and the tools that are currently being developed, such as using artificial intelligence (AI) to identify genetic mutations from histomorphological criteria. In summary, we find that the molecular genetic characterisation of canine neoplasms has made a promising start. As we understand more of the genetics underlying these tumours and more targeted therapies become available, it will no doubt become a mainstay in the delivery of precision veterinary care to dogs with cancer.
Digital PCR is a robust PCR technique that enables precise and accurate absolute quantitation of target molecules by diluting and partitioning of the samples into numerous compartments. Automated partitioning can be attained by creating “water-in-oil” emulsion (emulsion-based digital PCR) or using a chip with microchannels (microfluidics-based digital PCR). We discuss the advantages and a wide variety of clinical applications of this technique. We describe the droplet digital RT-PCR protocol published by Jennings et al. for identification and absolute quantitation of BCR-ABL1 transcripts.
Real-time PCR used to be the gold standard when it comes to detection of rare mutations, copy number variations or genetically modified organisms. A new option for DNA analyses is the digital PCR. In digital PCR, the reaction mix is distributed to many partitions and endpoint PCR is performed. The fraction of positive partitions can be used to calculate the initial concentration. Digital PCR is highly sensitive and allows quantification of absolute copy numbers without using a standard curve.
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