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BMC Genomics Jun 2024Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied...
BACKGROUND
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
RESULTS
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
CONCLUSION
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.
Topics: Exons; Software; DNA Primers; Internet; Humans; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 38867172
DOI: 10.1186/s12864-024-10456-2 -
Molecules (Basel, Switzerland) Oct 2019Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics... (Review)
Review
Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.
Topics: Aptamers, Nucleotide; DNA Primers; DNA, Single-Stranded; Gene Library; High-Throughput Nucleotide Sequencing; Nucleic Acid Amplification Techniques; Nucleic Acids; Polymerase Chain Reaction; SELEX Aptamer Technique
PubMed: 31591283
DOI: 10.3390/molecules24193598 -
Analytical Biochemistry Feb 2017DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention...
DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention in recent years. DNA extracted from these samples is often inapplicable for conventional polymerase chain reaction (PCR), so for its amplification the nearby primers are commonly used. Here we report the data that clarify the features of PCR with nearby and abutting primers. We have shown that the proximity of primers leads to significant reduction of the reaction time and ensures the successful performance of DNA amplification even in the presence of PCR inhibitors. The PCR with abutting primers is usually characterized by the absence of nonspecific amplification products that causes extreme sensitivity with limit of detection on single copy level. The feasibility of PCR with abutting primers was demonstrated on species identification of 100 years old rotten wood.
Topics: DNA Primers; DNA, Plant; Plants; Polymerase Chain Reaction
PubMed: 27908595
DOI: 10.1016/j.ab.2016.11.017 -
Analytical and Bioanalytical Chemistry Nov 2022A simple, sensitive, specific and fast method based on the loop-mediated isothermal amplification (LAMP) technique and cleavable molecular beacon (CMB) was developed for...
A simple, sensitive, specific and fast method based on the loop-mediated isothermal amplification (LAMP) technique and cleavable molecular beacon (CMB) was developed for chicken authentication detection. LAMP and CMB were used for DNA amplification and amplicon analysis, respectively. Targeting the mitochondrial cytochrome b gene of chickens, five primers and one CMB probe were designed, and their specificity was validated against nine other animal species. The structure of CMB and concentrations of dNTPs, MgSO, betaine, RNase H2, primers and CMB were optimized. The CMB-LAMP assay was completed within 17 min, and its limit of detection for chicken DNA was 1.5 pg μL. Chicken adulteration as low as 0.5% was detected in beef, and no cross-reactivity was observed. Finally, this assay was successfully applied to 20 commercial meat products. When combined with our developed DNA extraction method (the extraction time was 1 min: lysis for 10 s, washing for 20 s and elution for 30 s), the entire process (from DNA extraction to results analysis) was able to be completed within 20 min, which is at least 10 min shorter than other LAMP-based methods. Our method showed great potential for the on-site detection of chicken adulteration in meat.
Topics: Cattle; Animals; Chickens; Nucleic Acid Amplification Techniques; Meat; DNA Primers; DNA; Sensitivity and Specificity
PubMed: 36152037
DOI: 10.1007/s00216-022-04342-7 -
Applied Microbiology and Biotechnology Dec 2014The identification and quantification of specific organisms in mixed microbial communities often relies on the ability to design oligonucleotide probes and primers with... (Review)
Review
The identification and quantification of specific organisms in mixed microbial communities often relies on the ability to design oligonucleotide probes and primers with high specificity and sensitivity. The design of these oligonucleotides (or "oligos" for short) shares many of the same principles in spite of their widely divergent applications. Three common molecular biology technologies that require oligonucleotide design are polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), and DNA microarrays. This article reviews techniques and software available for the design and optimization of oligos with the goal of targeting a specific group of organisms within mixed microbial communities. Strategies for enhancing specificity without compromising sensitivity are described, as well as design tools well suited for this purpose.
Topics: Biota; Computer-Aided Design; DNA Primers; In Situ Hybridization, Fluorescence; Metagenomics; Microarray Analysis; Models, Theoretical; Oligonucleotide Probes; Polymerase Chain Reaction; Software
PubMed: 25359473
DOI: 10.1007/s00253-014-6165-x -
Genes Sep 2021PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a...
PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting synthesis of DNA primers, and especially when selecting the 3'nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3'-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs ddNTPs at the 3'elongation site, thus avoiding synthesis of abortive primers.
Topics: Amino Acid Sequence; DNA Primase; DNA Primers; DNA Replication; DNA, Mitochondrial; DNA-Directed DNA Polymerase; Dideoxynucleotides; Humans; Multifunctional Enzymes; Nucleotides
PubMed: 34680882
DOI: 10.3390/genes12101487 -
Briefings in Bioinformatics May 2022Primers are critical for polymerase chain reaction (PCR) and influence PCR experimental outcomes. Designing numerous combinations of forward and reverse primers involves...
Primers are critical for polymerase chain reaction (PCR) and influence PCR experimental outcomes. Designing numerous combinations of forward and reverse primers involves various primer constraints, posing a computational challenge. Most PCR primer design methods limit parameters because the available algorithms use general fitness functions. This study designed new fitness functions based on user-specified parameters and used the functions in a primer design approach based on the multiobjective particle swarm optimization (MOPSO) algorithm to address the challenge of primer design with user-specified parameters. Multicriteria evaluation was conducted simultaneously based on primer constraints. The fitness functions were evaluated using 7425 DNA sequences and compared with a predominant primer design approach based on optimization algorithms. Each DNA sequence was run 100 times to calculate the difference between the user-specified parameters and primer constraint values. The algorithms based on fitness functions with user-specified parameters outperformed the algorithms based on general fitness functions for 11 primer constraints. Moreover, MOPSO exhibited superior implementation in all experiments. Practical gel electrophoresis was conducted to verify the PCR experiments and established that MOPSO effectively designs primers based on user-specified parameters.
Topics: Algorithms; Base Sequence; DNA Primers; Polymerase Chain Reaction; Software
PubMed: 35397164
DOI: 10.1093/bib/bbac121 -
The Analyst Jan 2020Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of...
Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3' end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 10 DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.
Topics: DNA; DNA Primers; Fluorescence Resonance Energy Transfer; Nucleic Acid Amplification Techniques; Polymorphism, Single Nucleotide; Recombinases; Thermus thermophilus
PubMed: 31793929
DOI: 10.1039/c9an01701a -
Molecular Ecology Resources Jan 2016The arc of reception for a new technology or method--like the reception of new information itself--can pass through predictable stages, with audiences' responses...
The arc of reception for a new technology or method--like the reception of new information itself--can pass through predictable stages, with audiences' responses evolving from 'I don't believe it', through 'well, maybe' to 'yes, everyone knows that' to, finally, 'old news'. The idea that one can sample a volume of water, sequence DNA out of it, and report what species are living nearby has experienced roughly this series of responses among biologists, beginning with the microbial biologists who developed genetic techniques to reveal the unseen microbiome. 'Macrobial' biologists and ecologists--those accustomed to dealing with species they can see and count--have been slower to adopt such molecular survey techniques, in part because of the uncertain relationship between the number of recovered DNA sequences and the abundance of whole organisms in the sampled environment. In this issue of Molecular Ecology Resources, Evans et al. (2015) quantify this relationship for a suite of nine vertebrate species consisting of eight fish and one amphibian. Having detected all of the species present with a molecular toolbox of six primer sets, they consistently find DNA abundances are associated with species' biomasses. The strength and slope of this association vary for each species and each primer set--further evidence that there is no universal parameter linking recovered DNA to species abundance--but Evans and colleagues take a significant step towards being able to answer the next question audiences tend to ask: 'Yes, but how many are there?'
Topics: Animals; DNA; DNA Primers; Ecology; Fishes
PubMed: 26768195
DOI: 10.1111/1755-0998.12455 -
ACS Synthetic Biology May 2022With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications including gene synthesis,...
With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications including gene synthesis, CRISPR-based gene editing, and DNA data storage. The selective retrieval of specific oligos from these complex pools traditionally uses polymerase chain reaction (PCR). Designing a large number of primers to use in PCR presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool is orders of magnitude larger than other applications. Although a nested primer address system was recently developed to increase the number of accessible files for DNA storage, it requires more complicated lab protocols and more expensive reagents to achieve high specificity, as well as more DNA address space. Here, we present a new combinatorial PCR method that has none of those drawbacks and outperforms in retrieval specificity. In experiments, we accessed three files that each comprised 1% of a DNA prototype database that contained 81 different files and enriched them to over 99.9% using our combinatorial primer method. Our method provides a viable path for scaling up DNA data storage systems and has broader utility whenever one must access a specific target oligo and can design their own primer regions.
Topics: DNA; DNA Primers; Databases, Nucleic Acid; Information Storage and Retrieval; Polymerase Chain Reaction
PubMed: 35191684
DOI: 10.1021/acssynbio.1c00482