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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 -
Methods in Molecular Biology (Clifton,... 2023PCR allele competitive extension (PACE™) is a marker system to analyze single nucleotide polymorphisms (SNPs). As strongly related technologies like kompetitive...
PCR allele competitive extension (PACE™) is a marker system to analyze single nucleotide polymorphisms (SNPs). As strongly related technologies like kompetitive allele-specific PCR (KASP™) or Amplifluor, PACE is based on a polymerase chain reaction (PCR) with two competing allele-specific primers and an endpoint fluorescent measurement. The system requires sequence information surrounding the SNP of interest and a qPCR machine or a fluorescent plate reader.
Topics: Genotype; Polymerase Chain Reaction; Alleles; DNA Primers; Polymorphism, Single Nucleotide; Coloring Agents
PubMed: 36781648
DOI: 10.1007/978-1-0716-3024-2_18 -
BMC Microbiology Jun 2020Repetitive-PCR (rep-PCR) using BOXA1R and BOXA2R as single primers was investigated for its potential to genotype bacteriophage. Previously, this technique has been...
BACKGROUND
Repetitive-PCR (rep-PCR) using BOXA1R and BOXA2R as single primers was investigated for its potential to genotype bacteriophage. Previously, this technique has been primarily used for the discrimination of bacterial strains. Reproducible DNA fingerprint patterns for various phage types were generated using either of the two primers.
RESULTS
The similarity index of replicates ranged from 89.4-100% for BOXA2R-PCR, and from 90 to 100% for BOXA1R-PCR. The method of DNA isolation (p = 0.08) and the phage propagation conditions at two different temperatures (p = 0.527) had no significant influence on generated patterns. Rep-PCR amplification products were generated from different templates including purified phage DNA, phage lysates and phage plaques. The use of this method enabled comparisons of phage genetic profiles to establish their similarity to related or unrelated phages and their bacterial hosts.
CONCLUSION
The findings suggest that repetitive-PCR could be used as a rapid and inexpensive method to preliminary screen phage isolates prior to their selection for more comprehensive studies. The adoption of this rapid, simple and reproducible technique could facilitate preliminary characterisation of a large number of phage isolates and the investigation of genetic relationship between phage genotypes.
Topics: Bacteriophages; DNA Primers; DNA, Viral; Genotyping Techniques; Phylogeny; Polymerase Chain Reaction; Temperature
PubMed: 32527227
DOI: 10.1186/s12866-020-01770-2 -
Methods in Molecular Biology (Clifton,... 2023Polymerase chain reaction (PCR) is a laboratory technique used to amplify a targeted region of DNA, demarcated by a set of oligonucleotide primers. Long-range PCR is a...
Polymerase chain reaction (PCR) is a laboratory technique used to amplify a targeted region of DNA, demarcated by a set of oligonucleotide primers. Long-range PCR is a form of PCR optimized to facilitate the amplification of large fragments. Using the adapted long-range PCR protocol described in this chapter, we were able to generate PCR products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples. For some of the long PCRs, successful amplification was not possible without the use of PCR enhancers. Thus, we also evaluated the impact of some enhancers on long-range PCR and included the findings as part of this updated chapter.
Topics: Humans; Polymerase Chain Reaction; DNA Primers; Data Collection
PubMed: 37608112
DOI: 10.1007/978-1-0716-3358-8_15 -
Methods in Molecular Biology (Clifton,... 2023Single-nucleotide polymorphisms (SNPs), the most abundant genetic variation in the population, have become the molecular marker of choice. Generally, the efficient...
Single-nucleotide polymorphisms (SNPs), the most abundant genetic variation in the population, have become the molecular marker of choice. Generally, the efficient detection of SNPs requires specialized costly equipment. Although there are a few strategies for detecting SNPs through polymerase chain reaction, followed by restriction enzyme digestion and agarose gel electrophoresis, these methods are time-consuming and might be less diagnostic. Interestingly, the tetra primer amplification refractory mutation system (T-ARMS) strategy utilizes a pair of allele-specific primers in a single PCR for the diagnostic detection of SNPs in a codominant manner through standard agarose gel electrophoresis. The simplicity and robustness of the strategy have inspired the researchers to adopt this low-cost method of SNP detection in different crop plants. Here, we have described the principle, methods, and conditions for the T-ARMS strategy. The described methodology starts from the isolation of genomic DNA and ends with the post-PCR analysis of refractory amplicons in standard agarose gel electrophoresis. The limitations and future perspectives are also discussed. Taken together, T-ARMS evolves as a method of choice for low-cost SNP detection in plants.
Topics: DNA Primers; Mutation; Polymerase Chain Reaction; DNA; Polymorphism, Single Nucleotide; Genotype
PubMed: 36781652
DOI: 10.1007/978-1-0716-3024-2_22 -
Nucleic Acids Research Jun 2022The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), synthesizes chimeric RNA-DNA primers of a limited length for DNA polymerases...
The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), synthesizes chimeric RNA-DNA primers of a limited length for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA-DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (∼35-mer): (i) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58C) with the primer 5'-end, and (ii) flexible tethering of p58C and the DNA polymerase alpha catalytic core domain (p180core) to the primosome platform domain by extended linkers. The obtained data allow us to conclude that p58C is a key regulator of all steps of RNA-DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.
Topics: Chromosomes; DNA; DNA Polymerase I; DNA Primase; DNA Primers; DNA Replication; DNA-Directed DNA Polymerase; Humans; RNA
PubMed: 35689638
DOI: 10.1093/nar/gkac492 -
Biosensors & Bioelectronics Apr 2021We herein describe a novel technology, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP), enabling target nucleic acid detection....
We herein describe a novel technology, termed self-priming phosphorothioated hairpin-mediated isothermal amplification (SP-HAMP), enabling target nucleic acid detection. Isothermal amplification strategies are a simple process that efficiently raises the amount of nucleic acid at a constant temperature, but still has lots of problems such as the requirement of multiple exogenous primers and enzymes, which trigger non-specific background signal and increase the complexity of procedures. The key component for overcoming the above-mentioned limitations is the designed hairpin probe (HP) consisting of self-priming region along the 3' stem and the 3' overhang and phosphorothioate modifications at the 5' overhang and the specific loop part. The HP was designed to open through binding to target nucleic acid. Upon opening of HP, its self-priming (SP) region is rearranged to form a smaller hairpin whose 3' end could serve as a primer. The following extension produces the extended HP and displaces the bound target nucleic acid, which is then recycled to open another HP. Due to the reduced stability caused by the specific two phosphorothioate (PS) modifications, the 3' end of EP1 is readily rearranged to form the foldback hairpin structure, which would promote the foldback extension to produce once more extended HP. Since the two PS modifications are always located at the same positions along the 5' stem within the further extended HPs, the foldback reaction followed by the extension would be continuously repeated, consequently producing a large number of the long hairpin concatamers. Based on this unique design principle, we successfully detected even a single copy of target DNA with outstanding discrimination capability under an isothermal condition by employing only a single HP without the requirement for the complicated multiple primers. In conclusion, the sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling short target nucleic acid detection such as microRNAs or any target which is less than 200 mer.
Topics: Biosensing Techniques; DNA; DNA Primers; Nucleic Acid Amplification Techniques; Nucleic Acids
PubMed: 33548651
DOI: 10.1016/j.bios.2021.113051 -
Molecules (Basel, Switzerland) May 2023Honey is a widely consumed natural product, and its entomological origin can significantly influence its market value. Therefore, traceability of the entomological... (Review)
Review
Honey is a widely consumed natural product, and its entomological origin can significantly influence its market value. Therefore, traceability of the entomological origin of honey should also be considered in honey quality control protocols. Although several methods exist, such as physicochemical characterization and bioactivity profiling of honey of different entomological origins, the most promising three methods for entomological authentication of honey include protein-based identification, chemical profiling, and a DNA-based method. All of these methods can be applied for reliable identification of the entomological origin of honey. However, as the honey is a complex matrix, the inconsistency of the results obtained by these methods is a pragmatic challenge, and therefore, the use of each method in all the cases is questionable. Most of these methodologies can be used for authentication of newly harvested honey and it is worth understanding the possibility of using these methods for authentication of relatively old samples. Most probably, using DNA-based methods targeting small fragments of DNA can provide the best result in old samples, however, the species-specific primers targeting short fragments are limited and not available for all species. Therefore, using universal primers in combination with a DNA metabarcoding approach can be a good solution that requires further investigation. This present article describes the applications of different methods, their pros, and their cons to identify honey based on entomological origin.
Topics: Honey; DNA; DNA Primers; Species Specificity; Biological Products
PubMed: 37241972
DOI: 10.3390/molecules28104232 -
The Journal of Molecular Diagnostics :... Nov 2022Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the...
Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.
Topics: Humans; Taq Polymerase; Alleles; DNA; DNA Primers; Polymerase Chain Reaction
PubMed: 36058471
DOI: 10.1016/j.jmoldx.2022.08.002 -
Applied Microbiology and Biotechnology Sep 2022Micropipette tips are currently among the most used disposable devices in bioresearch and development laboratories. Their main application is the fractionation of...
Micropipette tips are currently among the most used disposable devices in bioresearch and development laboratories. Their main application is the fractionation of solutions. New functionalities have recently been added to this device, widening their applications. This paper analyzed disposable micropipette tips as reagent holders of PCR reagents. PCR has become a prevalent and often indispensable technique in biological laboratories for various applications, such as the detection of coronavirus and other infectious diseases. A functional micropipette tip was implemented to simplify PCR analysis and reduce the contamination chances of deoxynucleotides and specific primers. This disposable device is prepared by tip coating processes of reagents, using polyvinyl alcohol polymer and additives. The coated layer is optimized to load and release PCR reagents efficiently. As a proof of concept, we show that the detection of Bordetella pertussis, the etiological agent of whooping cough whose diagnostic relies on PCR, can be quickly done using practical-functional tips. This device is an excellent example of testing the functionality and contribution of molecular diagnostic PCR tips. KEY POINTS: • Functional micropipette tips are prepared by coating with dNTPs and primers. • Functional tips are used to replace dNTPs and primers in the PCR master mix. • PCR diagnostic of Bordetella pertussis is performed using functional tips.
Topics: Bordetella pertussis; DNA Primers; DNA, Bacterial; Humans; Polymerase Chain Reaction; Whooping Cough
PubMed: 35915170
DOI: 10.1007/s00253-022-12069-9