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Molecular Ecology Resources May 2022Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of... (Review)
Review
Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during "library preparation", that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one-step PCR, two-step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.
Topics: Biodiversity; DNA Barcoding, Taxonomic; DNA Primers; Gene Library; Polymerase Chain Reaction
PubMed: 34551203
DOI: 10.1111/1755-0998.13512 -
Experimental Parasitology Mar 2022Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating...
Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/μl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.
Topics: Centrifugation; Cryptosporidium; DNA Primers; DNA, Protozoan; Filtration; Limit of Detection; Polymerase Chain Reaction; Sensitivity and Specificity; Wastewater
PubMed: 35104468
DOI: 10.1016/j.exppara.2022.108216 -
Molecules (Basel, Switzerland) Jun 2023This study aimed to explore the applicability of DNA barcoding for assessing the authenticity of caviar on the Chinese market. A set of universal primers and two sets...
This study aimed to explore the applicability of DNA barcoding for assessing the authenticity of caviar on the Chinese market. A set of universal primers and two sets of designed primers based on and genes were used to identify maternal species of samples from 21 batches of caviar. The results showed that the PCR products from three sets of primers had more than 98% similarity to the sequences in database. The gene could not distinguish sturgeons with closed genetic relationships, while gene could effectively improve the accuracy of DNA barcoding and was more suitable to the identification of interspecific sturgeon than the gene. The neighbor-joining dendrogram further confirmed the applicability and accuracy of and genes in identifying maternal relatives of caviar (////. Despite the limitations of mitochondrial DNA in identifying hybrid sturgeon species, the presence of counterfeit caviar of non-sturgeon ingredients could be excluded. All the caviar samples were identified successfully as sturgeon species, but the mislabeling rate of species was 33.4%, indicating that there were illegal phenomena such as disorderly labeling, mislabeling, and adulteration on the market.
Topics: Animals; DNA Barcoding, Taxonomic; DNA, Mitochondrial; Fishes; Polymerase Chain Reaction; DNA Primers
PubMed: 37446706
DOI: 10.3390/molecules28135046 -
Molecular Ecology Resources Jan 2023Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially... (Review)
Review
Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially reduced data output due to the disproportionate amplification of the DNA of the focal predator, here termed "the predator problem". We review the various methods commonly used to overcome this problem, from deeper sequencing to exclusion of predator DNA during PCR, and how they may interfere with increasingly common multipredator-taxon studies. We suggest that multiprimer approaches with an emphasis on achieving both depth and breadth of prey detections may overcome the issue to some extent, although multitaxon studies require further consideration, as highlighted by an empirical example. We also review several alternative methods for reducing the prevalence of predator DNA that are conceptually promising but require additional empirical examination. The predator problem is a key constraint on molecular dietary analyses but, through this synthesis, we hope to guide researchers in overcoming this in an effective and pragmatic way.
Topics: Animals; Food Chain; Predatory Behavior; DNA Primers; Polymerase Chain Reaction; DNA; Diet
PubMed: 36017818
DOI: 10.1111/1755-0998.13705 -
Methods in Molecular Biology (Clifton,... 2022The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties. The software provides...
The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties. The software provides comprehensive tools for designing primers for most PCR and perspective applications, including standard, multiplex, long-distance, inverse, real-time with TaqMan probe, Xtreme Chain Reaction (XCR), group-specific, overlap extension PCR for multifragment assembling cloning, and isothermal amplification (Loop-mediated Isothermal Amplification). A program is available to design specific oligonucleotide sets for long sequence assembly by ligase chain reaction and to design multiplexed of overlapping and nonoverlapping DNA amplicons that tile across a region(s) of interest for targeted next-generation sequencing, competitive allele-specific PCR (KASP)-based genotyping assay for single-nucleotide polymorphisms and insertions and deletions at specific loci, among other features. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. FastPCR includes various bioinformatics tools for analysis and searching of sequences, restriction I-II-III-type enzyme endonuclease analysis, and pattern searching. The program also supports the assembly of a set of contiguous sequences, consensus sequence generation, and sequence similarity and conservancy analysis. FastPCR performs efficient and complete detection of various repeat types with visual display. FastPCR allows for sequence file batch processing that is essential for automation. The software is available for download at https://primerdigital.com/fastpcr.html and online version at https://primerdigital.com/tools/pcr.html .
Topics: Computer Simulation; DNA Primers; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Oligonucleotides; Polymerase Chain Reaction; Software
PubMed: 34773626
DOI: 10.1007/978-1-0716-1799-1_16 -
Talanta Jan 2021Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential...
Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies. Despite these advantages, ePCR remains underused due to the lack of optimal starting conditions, straightforward methods to evaluate success, and guidelines for tuning the reaction. This knowledge has been elusive for bulk emulsion generation methods, such as stirring and vortexing, the only methods that can emulsify libraries of ≥10 sequences within minutes, because these emulsions have not been characterized in ways that preserve the heterogeneity that defines successful ePCR. Our study quantifies the outcome of ePCR from conditions specified in the literature using single particle analysis, which preserves this heterogeneity. We combine ePCR with magnetic microbeads and quantify the amplification yield via qPCR and the proportion of clonal and saturated beads via flow cytometry. Our single particle level analysis of thousands of beads resolves two key criteria that define the success of ePCR: 1) whether the target fraction of 20% clonal beads predicted by the Poisson distribution is achieved, and 2) whether those beads are partially or maximally covered by amplified DNA. We found that among the two concentrations of polymerase tested, only the higher one, which is 20-fold more than the concentration recommended for conventional PCR, could yield sufficient PCR products. Dramatic increases in the concentrations of reverse primer and nucleotides recommended in literature gave no measurable change in outcome. We thus provide evidence-based starting conditions for effective and economical ePCR for real DNA libraries and a straightforward workflow for evaluating the success of tuning ePCR prior to downstream applications.
Topics: DNA Primers; Emulsions; Gene Library; Polymerase Chain Reaction; Single Molecule Imaging
PubMed: 33076127
DOI: 10.1016/j.talanta.2020.121593 -
Nucleic Acids Research Nov 2022DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during...
DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.
Topics: Humans; DNA Polymerase I; Mutagens; DNA Replication; DNA Primase; Mutagenesis; DNA; DNA Primers; RNA
PubMed: 36454017
DOI: 10.1093/nar/gkac1101 -
PloS One 2023The rapid identification of Influenza A virus and its variants, which cause severe respiratory diseases, is imperative to providing timely treatment and improving...
A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan.
The rapid identification of Influenza A virus and its variants, which cause severe respiratory diseases, is imperative to providing timely treatment and improving patient outcomes. Conventionally, two separate assays (total test duration of up to 6 h) are required to initially differentiate Influenza A and B viruses and subsequently distinguish the pdm H1N1 and H3N2 serotypes of Influenza A virus. In this study, we developed a multiplex real-time RT-PCR method for simultaneously detecting Influenza A and B viruses and subtyping Influenza A virus, with a substantially reduced test duration. Clinical specimens from hospitalized patients and outpatients with influenza-like symptoms in Eastern Taiwan were collected between 2011 and 2015, transported to Hualien Tzu Chi Hospital, and analyzed. Conventional RT-PCR was used to subtype the isolated Influenza A viruses. Thereafter, for rapid identification, the multiplex real-time RT-PCR method was developed and applied to identify the conserved regions that aligned with the available primers and probes. Accordingly, a multiplex RT-PCR assay with three groups of primers and probes (MAF and MAR primers and MA probe; InfAF and InfAR primers and InfA probe; and MBF and MBR primers and MB probe) was established to distinguish these viruses in the same reaction. Thus, with this multiplex RT-PCR assay, Influenza B, Influenza A pdm H1N1, and Influenza A H3N2 viruses were accurately detected and differentiated within only 2.5 h. This multiplex RT-PCR assay showed similar analytical sensitivity to the conventional singleplex assay. Further, the phylogenetic analyses of our samples revealed that the characteristics of these viruses were different from those reported previously using samples collected during 2012-2013. In conclusion, we developed a multiplex real-time RT-PCR method for highly efficient and accurate detection and differentiation of Influenza A and B viruses and subtyping Influenza A virus with a substantially reduced test duration for diagnosis.
Topics: Humans; Influenza, Human; Influenza A Virus, H3N2 Subtype; Influenza A Virus, H1N1 Subtype; Taiwan; Phylogeny; Sensitivity and Specificity; Influenza A virus; Mutation; DNA Primers; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 36952488
DOI: 10.1371/journal.pone.0283074 -
Nucleosides, Nucleotides & Nucleic Acids 2020Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of...
Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of primers is one of the main determinants affecting specificity, sensitivity and reliability of the reaction. Here, we report on the results of the detailed study devoted to the dimerization of the primers during PCR. The course and specificity of the reaction were studied on the model DNA templates as well as genomic DNA using primers that form amplifiable heterodimeric structures with different thermodynamic stability. It was confirmed that more than two 3'-overlapping nucleotides cause a considerable accumulation of primer dimers. It turned out that the presence of any DNA promotes the formation of dimers even for primers, which do not tend to nonspecific amplification in the absence of DNA. It was shown that dimerization could not be eliminated by commonly used techniques. Even the use of hot-start DNA polymerases does not prevent PD formation if primers with stable 3'-overlapping are employed. Despite several advantages of PCR with abutting primers, their close disposition has no benefits regarding the formation of PD if low-quality primers are utilized.
Topics: DNA; DNA Primers; Dimerization; Polymerase Chain Reaction; Thermodynamics
PubMed: 32799617
DOI: 10.1080/15257770.2020.1803354 -
Chembiochem : a European Journal of... Apr 2024Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an... (Review)
Review
Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an aqueous, protecting group free system, without the need for expensive enzymes. However, these primer extension reactions suffer from a lack of fidelity, low reaction rates, low overall yields, and short primer extension lengths. This review outlines a detailed mechanistic pathway for non-enzymatic template-directed primer extension and presents a review of the thermodynamic driving forces involved in entropic templating. Through the lens of entropic templating, the rate and fidelity of a reaction are shown to be intrinsically linked to the reactivity of the activating agent used. Thus, a strategy is discussed for the optimization of non-enzymatic template-directed primer extension, providing a path towards cost-effective in vitro synthesis of RNA and DNA.
Topics: DNA Primers; Nucleic Acids; DNA; RNA; Thermodynamics; Templates, Genetic
PubMed: 38282207
DOI: 10.1002/cbic.202300859