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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,... 2015
Topics: DNA Primers; Polymerase Chain Reaction
PubMed: 25848662
DOI: 10.1007/978-1-4939-2365-6 -
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 -
Advances in Experimental Medicine and... 2017This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA... (Review)
Review
This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.
Topics: Animals; DNA; DNA Polymerase I; DNA Primase; DNA Primers; DNA Replication; Eukaryota; Eukaryotic Cells; Humans; Kinetics; RNA
PubMed: 29357056
DOI: 10.1007/978-981-10-6955-0_6 -
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 -
The Enzymes 2019PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA... (Review)
Review
PrimPol is the second primase discovered in eukaryotic cells, whose function is to restart the stalled replication forks during both mitochondrial and nuclear DNA replication. This chapter revises our current knowledge about the mechanism of synthesis of DNA primers by human PrimPol, and the importance of its distinctive Zn-finger domain (ZnFD). After PrimPol forms a binary complex with ssDNA, the formation of the pre-ternary complex strictly requires the presence of Mn ions to stabilize the interaction of the incoming deoxynucleotide at the 3'-site. The capacity to bind both ssDNA template and 3'-deoxynucleotide was shown to reside in the AEP core of PrimPol, with ZnFD being dispensable at these two early steps of the primase reaction. Sugar selection favoring dNTPs versus NTPs at the 3' site is mediated by a specific tyrosine (Tyr) acting as a steric gate. Besides, a specific glutamate residue (Glu) conforming a singular A motif (DxE) promotes the use of Mn to stabilize the pre-ternary complex. Mirroring the function of the PriL subunit of dimeric AEP primases, the ZnFD of PrimPol is crucial to stabilize the initiating 5'-nucleotide, specifically interacting with the gamma-phosphate. Such an interaction is crucial to optimize dimer formation and the subsequent translocation events leading to the processive synthesis of a mature DNA primer. Finally, the capacity of PrimPol to tolerate lesions is discussed in the context of its DNA primase function, and its potential as a TLS primase.
Topics: DNA Primase; DNA Primers; DNA Replication; DNA-Directed DNA Polymerase; Humans; Multifunctional Enzymes
PubMed: 31627881
DOI: 10.1016/bs.enz.2019.06.003 -
Current Protocols in Human Genetics Apr 2019Mapping patterns of DNA methylation throughout the epigenome are critical to our understanding of several important biological and regulatory functions, such as...
Mapping patterns of DNA methylation throughout the epigenome are critical to our understanding of several important biological and regulatory functions, such as transcriptional regulation, genomic imprinting, and embryonic development. The development and rapid advancement of next-generation sequencing (NGS) technologies have provided clinicians and researchers with accurate and reliable read-outs of genomic and epigenomic information at the nucleotide level. Such improvements have significantly lowered the cost required for genome-wide sequencing, facilitating the vast acquisition of data that has led to many improvements in patient care. However, the torrid rate of NGS data generation has left targeted validation approaches behind, including the confirmation of epigenetic marks such as DNA methylation. To overcome these shortcomings, we present a rapid and robust protocol for the parallel examination of multiple methylated sequences that we have termed simultaneous targeted methylation sequencing (sTM-Seq). Key features of this technique include the elimination of the need for large amounts of high-molecular weight DNA and the nucleotide specific distinction of both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Moreover, sTM-Seq is scalable and can be used to investigate multiple loci in dozens of samples within a single sequencing run. By utilizing freely available web-based software and universal primers for multipurpose barcoding, library preparation, and customized sequencing, sTM-Seq is affordable, efficient, and widely applicable. Together, these features enable sTM-Seq to have wide-reaching clinical applications that will greatly improve turnaround rates for same-day procedures and allow clinicians to collect high-resolution data that can be used in a variety of patient settings. © 2019 by John Wiley & Sons, Inc.
Topics: 5-Methylcytosine; DNA; DNA Methylation; DNA Primers; Epigenesis, Genetic; Genome; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA; Software
PubMed: 30620135
DOI: 10.1002/cphg.81 -
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