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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 -
The Science of the Total Environment Sep 2020Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the nitrogen cycle by coupling ammonium and nitrite to produce dinitrogen gas (N). Polymerase... (Review)
Review
Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the nitrogen cycle by coupling ammonium and nitrite to produce dinitrogen gas (N). Polymerase chain reaction (PCR) is a fast, simple, and sensitive method that is widely used to assess the diversity, abundance, and activity of the slow-growing bacteria. In this review, we summarize and evaluate the wide variety of PCR primers targeting the 16S rRNA gene and functional genes (hzo, nir, and hzs) of anammox bacteria for their effectiveness and efficiencies in detecting this group of bacteria in different sample types. Furthermore, the efficiencies of different universal high-throughput sequencing 16S rRNA gene primers in anammox bacteria investigations were also evaluated to provide a reference for primer selection. Based on our in silico evaluation results, none of the 16S rRNA gene primers could recover all of the known anammox bacteria, but multiple hzo and hzs gene primers could accomplish this task. However, uncertain copies (1-3 copies) of hzo genes were identified in the genomes, and the hydrazine oxidation reaction catalyzed by hydrazine oxidoreductases (HZOs) can also be catalyzed by other hydroxylamine oxidoreductases (HAOs) in anammox bacteria, which can potentially result in large deviations in hzo-based qPCR and RT-qPCR analyses and results. Therefore, the use of optimal primers targeting unique hzs genes are recommended, although the efficiencies of these newly designed primers need further verification in practical applications. This article provides comprehensive information for the effective and specific detection of anammox bacteria using specific primers targeting the 16S rRNA gene and functional genes and serves as a basis for future high-quality primer design.
Topics: Bacteria; DNA Primers; DNA, Bacterial; Oxidation-Reduction; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 16S
PubMed: 32460079
DOI: 10.1016/j.scitotenv.2020.139387 -
Journal of Microbiological Methods Aug 2020Appropriate use and specific primers are important in assessing the diversity and abundance of microbial groups of interest. Bdellovibrio and like organisms (BALOs),...
Appropriate use and specific primers are important in assessing the diversity and abundance of microbial groups of interest. Bdellovibrio and like organisms (BALOs), that refer to obligate Gram-negative bacterial predators of other Gram-negative bacteria, evolved in terms of taxonomy and classification over the past two decades. Hence, some former primers have become inadequate while others are yet to be designed, for both PCR (especially with the advent of NGS) and qPCR approaches. Thus, to study BALOs' abundance and diversity in a variety of aquatic ecosystems, we designed in silico specific primer sets for each BALO genera and tested them in vitro on a variety of cultures and environmental samples. Also, we performed Sanger and Nano Miseq sequencing to reveal the exact degree of specificity of the most promising primers set. Here we report our success in designing specific primers for some BALOs genera, i.e. Bdellovibrio (PCR), Bacteriovorax (qPCR), Peredibacter (PCR).
Topics: Bdellovibrio; DNA Primers; DNA, Bacterial; DNA, Ribosomal; Ecosystem; Environmental Monitoring; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Water Microbiology
PubMed: 32598974
DOI: 10.1016/j.mimet.2020.105996 -
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 -
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 -
ACS Applied Materials & Interfaces May 2023Due to its high coding density and longevity, DNA is a compelling data storage alternative. However, current DNA data storage systems rely on the de novo synthesis of...
Due to its high coding density and longevity, DNA is a compelling data storage alternative. However, current DNA data storage systems rely on the de novo synthesis of enormous DNA molecules, resulting in low data editability, high synthesis costs, and restrictions on further applications. Here, we demonstrate the programmable assembly of reusable DNA blocks for versatile data storage using the ancient movable type printing principle. Digital data are first encoded into nucleotide sequences in DNA hairpins, which are then synthesized and immobilized on solid beads as modular DNA blocks. Using DNA polymerase-catalyzed primer exchange reaction, data can be continuously replicated from hairpins on DNA blocks and attached to a primer in tandem to produce new information. The assembly of DNA blocks is highly programmable, producing various data by reusing a finite number of DNA blocks and reducing synthesis costs (∼1718 versus 3000 to 30,000 US$ per megabyte using conventional methods). We demonstrate the flexible assembly of texts, images, and random numbers using DNA blocks and the integration with DNA logic circuits to manipulate data synthesis. This work suggests a flexible paradigm by recombining already synthesized DNA to build cost-effective and intelligent DNA data storage systems.
Topics: DNA; Information Storage and Retrieval; DNA Primers; Printing; Printing, Three-Dimensional
PubMed: 37184884
DOI: 10.1021/acsami.3c01860 -
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 -
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