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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 -
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 -
Methods (San Diego, Calif.) May 2023DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs...
DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs phosphoramidite chemistry and yields unmodified DNA generally attached to a surface at the 3' terminus. Having a freely accessible 3'-OH instead of 5'-OH is desirable too, and being able to introduce nucleoside analogs in a combinatorial manner is highly relevant in the context of nucleic acid therapeutics and in aptamer research. Here, we describe an enzymatic approach to the synthesis of high-density DNA microarrays that can also contain chemical modifications. The method uses a standard DNA microarray, to which a DNA primer is covalently bound through photocrosslinking. The extension of the primer with a DNA polymerase yields double-stranded DNA but is also amenable to the incorporation of modified dNTPs. Further processing with T7 exonuclease, which catalyzes the degradation of DNA in a specific (5'→3') direction, results in template strand removal. Overall, the method produces surface-bound natural and non-natural DNA oligonucleotides, is applicable to commercial microarrays and paves the way for the preparation of combinatorial, chemically modified aptamer libraries.
Topics: Oligonucleotide Array Sequence Analysis; DNA-Directed DNA Polymerase; DNA; DNA Primers; DNA Replication; Oligonucleotides
PubMed: 37001684
DOI: 10.1016/j.ymeth.2023.03.006 -
Nature Communications Jun 2021CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins...
CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
Topics: Bacteria; Bacterial Proteins; Bacteroidetes; CRISPR-Associated Proteins; CRISPR-Cas Systems; Computational Biology; DNA Primase; DNA Primers; DNA-Directed DNA Polymerase; Dimerization; Escherichia coli; Gene Expression; Mutation; Phylogeny; Prokaryotic Cells; Recombinant Proteins; Ribonucleotides
PubMed: 34140468
DOI: 10.1038/s41467-021-23535-9 -
BMC Bioinformatics May 2022Designing oligonucleotide primers and probes is one of the key steps of various laboratory experiments such as multiplexed PCR or digital multiplexed ligation assays....
BACKGROUND
Designing oligonucleotide primers and probes is one of the key steps of various laboratory experiments such as multiplexed PCR or digital multiplexed ligation assays. When designing multiplexed primers and probes to complex, heterogeneous DNA data sets, an optimization problem can arise where the smallest number of oligonucleotides covering the largest diversity of the input dataset needs to be identified. Tools that provide this optimization in an efficient manner for large input data are currently lacking.
RESULTS
Here we present Prider, an R package for designing primers and probes with a nearly optimal coverage for complex and large sequence sets. Prider initially prepares a full primer coverage of the input sequences, the complexity of which is subsequently reduced by removing components of high redundancy or narrow coverage. The primers from the resulting near-optimal coverage are easily accessible as data frames and their coverage across the input sequences can be visualised as heatmaps using Prider's plotting function. Prider permits efficient design of primers to large DNA datasets by scaling linearly to increasing sequence data, regardless of the diversity of the dataset.
CONCLUSIONS
Prider solves a recalcitrant problem in molecular diagnostics: how to cover a maximal sequence diversity with a minimal number of oligonucleotide primers or probes. The combination of Prider with highly scalable molecular quantification techniques will permit an unprecedented molecular screening capability with immediate applicability in fields such as clinical microbiology, epidemic virus surveillance or antimicrobial resistance surveillance.
Topics: DNA Primers; Multiplex Polymerase Chain Reaction; Oligonucleotides; Software
PubMed: 35549665
DOI: 10.1186/s12859-022-04710-1 -
Genes Jun 2022Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia caused by runt-related transcription factor 2 () mutations. In addition to the regular...
Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia caused by runt-related transcription factor 2 () mutations. In addition to the regular missense, small or large fragment deletions are the common mutation types of . This study aimed to find the rules of deletions in . The clinical information of one Chinese CCD family was collected. Genomic DNA was extracted for whole-exome sequencing (WES). Bioinformatics analyzed the pathogenicity of the variants. Polymerase chain reaction (PCR) and Sanger sequencing were carried out using specific primers. RT-PCR and Q-PCR were also used to detect the mRNA level of . The CCD studies related with deletions in from 1999 to 2021 from HGMD and PubMed were collected and analyzed for the relationship between the phenotypes and the length of deleted fragments. The proband presented typical CCD features, including delayed closure of cranial sutures, clavicle dysplasia, abnormal teeth. WES, PCR with specific primers and Sanger sequencing revealed a novel heterozygous 90-kbp deletion in (NG_008020.2 g.103671~193943), which caused a substitution (p.Asn183Ile) and premature termination (p.Asp184*). In addition, the mRNA expression of was decreased by 75.5% in the proband. Herein, 31 types of deletions varying from 2 bp to 800 kbp or covering the whole gene of were compared and the significant phenotypic difference was not found among these deletions. The CCD phenotypes were related with the final effects of mutation instead of the length of deletion. WES has the defects in identifying large indels, and direct PCR with specific primers and Sanger sequencing could make up for the shortcoming.
Topics: Cleidocranial Dysplasia; Core Binding Factor Alpha 1 Subunit; DNA Primers; Heterozygote; Humans; Mutation, Missense; RNA, Messenger
PubMed: 35885911
DOI: 10.3390/genes13071128 -
Nature Communications Jan 2022Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then...
Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5'-RNA-DNA-3' hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide stochastically during elongation and that this switches the primase into a dedicated DNA synthetic mode polymerase.
Topics: DNA Primase; DNA Primers; DNA Replication; DNA, Archaeal; DNA-Directed DNA Polymerase; Fluorescence Polarization; Kinetics; Models, Biological; Nucleotides; Polymerization; RNA, Archaeal; Stochastic Processes
PubMed: 35064114
DOI: 10.1038/s41467-022-28093-2 -
Scientific Reports Oct 2020The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity...
The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.
Topics: Animals; Biodiversity; DNA Barcoding, Taxonomic; DNA Primers; DNA, Ribosomal; Databases, Genetic; Fishes; Gene Library; Mitochondria; Species Specificity
PubMed: 33087755
DOI: 10.1038/s41598-020-74902-3 -
Scientific Reports Jun 2022Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However,...
Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However, finding suitable genetic markers with robust species-level resolution and primers targeting a broad species range among parasitic helminths are some of the challenges faced. This study aimed to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth (nematodes, trematodes, cestodes) DNA metabarcoding. To demonstrate the robustness of the 12S and 16S rRNA genes for DNA metabarcoding, we determined the proportion of species successfully recovered using mock helminth communities without environment matrix and mock helminth communities artificially spiked with environmental matrices. The environmental matrices are human fecal material, garden soil, tissue, and pond water. Our results revealed the robustness of the mitochondrial rRNA genes, through the high sensitivity of the 12S rRNA gene, and the effectiveness of the 12S and 16S primers targeting platyhelminths. With the mitochondrial rRNA genes, a broad range of parasitc helminths were successfully detected to the species level. The potential of the mitochondrial rRNA genes for helminth DNA metabarcoding was demonstrated, providing a valuable gateway for future helminth DNA metabarcoding applications like helminth detection and biodiversity studies.
Topics: Animals; DNA Barcoding, Taxonomic; DNA Primers; DNA, Helminth; Genes, rRNA; Helminths; Humans; RNA, Ribosomal, 16S
PubMed: 35705676
DOI: 10.1038/s41598-022-14176-z -
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