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Current Protocols in Immunology Nov 2018Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at...
Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy. Since the development of PLA in 2002, it has been increasingly used to detect the interaction between two proteins with high sensitivity and specificity. It is a simple and sensitive technique to study protein-protein interaction in cells. © 2018 by John Wiley & Sons, Inc.
Topics: Antibodies; Biological Assay; DNA Primers; Humans; Microscopy, Fluorescence; Protein Interaction Mapping
PubMed: 30238640
DOI: 10.1002/cpim.58 -
Biochemistry and Molecular Biology... 2011Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process...
Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software.
Topics: Base Sequence; DNA Primers; Databases, Genetic; Fluorescent Dyes; Humans; Laboratories; Molecular Sequence Data; Online Systems; Polymerase Chain Reaction; Software; Teaching; Transition Temperature
PubMed: 21445907
DOI: 10.1002/bmb.20461 -
Current Issues in Molecular Biology 2014Real time-quantitative PCR (RT-qPCR) technology has revolutionized the detection landscape in every area of molecular biology. The fundamental basis of this technology... (Review)
Review
Real time-quantitative PCR (RT-qPCR) technology has revolutionized the detection landscape in every area of molecular biology. The fundamental basis of this technology has remained unchanged since its inception, however various modifications have enhanced the overall performance of this highly versatile technology. These improvements have ranged from changes in the individual components of the enzymatic reaction cocktail (polymerizing enzymes, reaction buffers, probes, etc.) to the detection system itself (instrumentation, software, etc.). The RT-qPCR technology currently available to researchers is more sensitive, faster and affordable than when this technology was first introduced. In this article, we summarize the developments of the last few years in RT-qPCR technology and nucleic acid amplification.
Topics: Animals; DNA Primers; DNA-Directed DNA Polymerase; Genes, Essential; Humans; Oligonucleotides; Protein Engineering; Real-Time Polymerase Chain Reaction; Reference Standards; Software
PubMed: 23562919
DOI: No ID Found -
PCR Methods and Applications Dec 1993
Topics: DNA Primers; Polymerase Chain Reaction; Software
PubMed: 8118394
DOI: 10.1101/gr.3.3.s30 -
Nature Communications Apr 2022One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of...
One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.
Topics: Algorithms; DNA Primers; Likelihood Functions; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 35410464
DOI: 10.1038/s41467-022-29500-4 -
Biochimica Et Biophysica Acta May 2010DNA primase synthesizes short RNA primers that replicative polymerases further elongate in order to initiate the synthesis of all new DNA strands. Thus, primase owes its... (Review)
Review
DNA primase synthesizes short RNA primers that replicative polymerases further elongate in order to initiate the synthesis of all new DNA strands. Thus, primase owes its existence to the inability of DNA polymerases to initiate DNA synthesis starting with 2 dNTPs. Here, we discuss the evolutionary relationships between the different families of primases (viral, eubacterial, archael, and eukaryotic) and the catalytic mechanisms of these enzymes. This includes how they choose an initiation site, elongate the growing primer, and then only synthesize primers of defined length via an inherent ability to count. Finally, the low fidelity of primases along with the development of primase inhibitors is described.
Topics: Amino Acid Sequence; Animals; DNA Primase; DNA Primers; Evolution, Molecular; Humans; Molecular Sequence Data; Sequence Homology, Amino Acid
PubMed: 19540940
DOI: 10.1016/j.bbapap.2009.06.011 -
PloS One 2023qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However...
AIMS
qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6.
METHODS
The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer.
RESULTS
Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition.
CONCLUSIONS
Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.
Topics: Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; DNA; DNA Primers; Genomics
PubMed: 37083935
DOI: 10.1371/journal.pone.0284538 -
Molecular Ecology Dec 2023Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different... (Review)
Review
Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different trophic levels, and to evaluate impacts on interspecific interactions. Molecular approaches such as DNA metabarcoding are boosting our ability to perform biodiversity inventories. Nevertheless, even though a few studies have recently attempted exhaustive reconstructions of communities, holistic assessments remain rare. The majority of metabarcoding studies published in the last years used just one or two markers and analysed a limited number of taxonomic groups. Here, we provide an overview of emerging approaches that can allow all-taxa biological inventories. Exhaustive biodiversity assessments can be attempted by combining a large number of specific primers, by exploiting the power of universal primers, or by combining specific and universal primers to obtain good information on key taxa while limiting the overlooked biodiversity. Multiplexes of primers, shotgun sequencing and capture enrichment may provide a better coverage of biodiversity compared to standard metabarcoding, but still require major methodological advances. Here, we identify the strengths and limitations of different approaches, and suggest new development lines that might improve broad scale biodiversity analyses in the near future. More holistic reconstructions of ecological communities can greatly increase the value of metabarcoding studies, improving understanding of the consequences of ongoing environmental changes on the multiple components of biodiversity.
Topics: Biodiversity; DNA; DNA Barcoding, Taxonomic; DNA Primers; Ecology
PubMed: 36762839
DOI: 10.1111/mec.16881 -
BMC Bioinformatics Feb 2017Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction...
BACKGROUND
Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity.
RESULTS
Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species.
CONCLUSIONS
The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/ .
Topics: DNA Primers; Genetic Markers; Genome, Protozoan; Internet; Leishmania; Polymerase Chain Reaction; User-Computer Interface
PubMed: 28187714
DOI: 10.1186/s12859-017-1485-3 -
BMC Biotechnology Jun 2011MicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important...
BACKGROUND
MicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings.
RESULTS
We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification.
CONCLUSIONS
MiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification.
Topics: DNA Primers; MicroRNAs; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Temperature
PubMed: 21702990
DOI: 10.1186/1472-6750-11-70