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BACKGROUND: Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. METHODS: This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. RESULTS: The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. CONCLUSIONS: The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics. [ABSTRACT FROM AUTHOR]

The development and characterization of a new enzyme reaction contribute to advancements in modern biotechnology. Here, we report a novel CIS (clamping-mediated incorporation of single-stranded DNA with concomitant DNA synthesis) reaction catalyzed by Taq polymerase. In the reaction, a single-stranded DNA (ssDNA) with 3′ Cs is attached with a preformed 3′ G-tail of double-stranded DNA (dsDNA); DNA syntheses starting from both 3′ ends result in the incorporation of ssDNA. A 3′ G-tail length of 3 nucleotides adequately supports this reaction, indicating that Taq polymerase can clump short Watson–Crick base pairs as short as three pairs and use them to initiate DNA polymerization. The reverse transcriptase from Molony murine leukemia virus catalyzes strand displacement synthesis and produces flapped-end DNA, whereas the reaction by Taq polymerase involves the nick translation. These new reaction properties may be beneficial for the development of new molecular tools applicable in various fields. Apart from its CIS reaction activity, we also report that Taq polymerase has the undesirable characteristic of removing 5' fluorescent labels from dsDNA. This characteristic may have compromised various experiments involving the preparation of fluorescently-labeled dsDNA by PCR for a long time. [ABSTRACT FROM AUTHOR]

Detecting mutations related to tumors holds immense clinical significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a challenge for the advancement of low-copy mutant ctDNA genotyping in cancer cases. To address this, a Taqman qPCR strategy to identify rare mutations at low variant allele fractions (VAFs) has been developed. This strategy combines mutant-specific primers with wild-specific blockers. Diverging from other blocker-mediated PCRs, which rely on primer-induced strand displacement or the use of modified oligos resistant to Taq polymerase, our innovation is built upon the cleavage of specific blockers by Taq polymerase. Given its unique design, which does not hinge on strand displacement or base modification, we refer to this novel method as unmodified-blocker cleavage PCR (UBC-PCR). Multiple experiments consistently confirmed that variant distinction was improved significantly by introduction of 5′ unmatched blockers into the reaction. Moreover, UBC-PCR successfully detected mutant DNA at VAFs as low as 0.01% across six different variant contexts. Multiplex UBC-PCR was also performed to identify a reference target and three mutations with a sensitivity of 0.01% VAFs in one single tube. In profiling the gene status from 12 lung cancer ctDNA samples and 22 thyroid cancer FNA DNA samples, UBC-PCR exhibited a 100% concordance rate with ddPCR and a commercial ARMS kit, respectively. Our work demonstrates that UBC-PCR can identify low-abundance variants with high sensitivity in multiplex reactions, independent of strand displacement and base modification. This strategy holds the potential to significantly impact clinical practice and precision medicine. [ABSTRACT FROM AUTHOR]

DNA polymerases are fundamental enzymes that play a crucial role in processing DNA with high fidelity and accuracy ensuring the faithful transmission of genetic information. The recognition of unnatural base pairs (UBPs) by polymerases, enabling their replication, represents a significant and groundbreaking discovery with profound implications for genetic expansion. Romesberg et al. examined the impact of DNA containing 2,6-dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN) UBPs bound to T. aquaticus DNA polymerase (Taq) through crystal structure analysis. Here, we have used polarizable and nonpolarizable classical molecular dynamics (MD) simulations to investigate the structural aspects and stability of Taq in complex with a DNA duplex including a DS–DN pair in the terminal 3′ and 5′ positions. Our results suggest that the flexibility of UBP-incorporated DNA in the terminal position is arrested by the polymerase, thus preventing fraying and mispairing. Our investigation also reveals that the UBP remains in an intercalated conformation inside the active site, exhibiting two distinct orientations in agreement with experimental findings. Our analysis pinpoints particular residues responsible for favorable interactions with the UBP, with some relying on van der Waals interactions while other on Coulombic forces. [ABSTRACT FROM AUTHOR]

Computational and high-throughput experimental methods predict thousands of potential quadruplex sequences (PQSs) in the human genome. Often these PQSs contain more than four G-runs, which introduce additional uncertainty into the conformational polymorphism of the G4 DNA. G4-specific ligands, which are currently being actively developed as potential anticancer agents or tools for studying G4 structures in genomes, may preferentially bind to specific G4 structures over the others that can be potentially formed in the extended G-rich genomic region. We propose a simple technique that identifies the sequences that tend to form G4 in the presence of potassium ions or a specific ligand. Thermostable DNA Taq-polymerase stop assay can detect the preferential position of the G4 –ligand binging within a long PQS-rich genomic DNA fragment. This technique was tested for four G4 binders PDS, PhenDC3, Braco-19, and TMPyP4 at three promoter sequences of MYC, KIT, and TERT that contain several PQSs each. We demonstrate that the intensity of polymerase pausing reveals the preferential binding of a ligand to particular G4 structures within the promoter. However, the strength of the polymerase stop at a specific site does not always correlate with the ligand-induced thermodynamic stabilization of the corresponding G4 structure. [ABSTRACT FROM AUTHOR]

We demonstrate that base mismatches of caspase-3 DNA sequences can be detected by surface plasmon resonance (SPR) following signal amplification by polymerase from Thermus aquaticus ( Taq). The concentration of magnesium ions and the respective dNTPs for polymerase binding to the oligonucleotides on the sensing surface were optimized. Taq polymerase binds to double-stranded DNA that is self-assembled on the gold surface of the biosensor to induce an SPR signal. Experiments are presented on the effect of Mg(II) and dNTP concentrations on the activity of the polymerase on the sensing surface. The detection limits are 50 pM, 0.1 nM, 0.7 nM, 7 nM, and 20 nM for correctly matched, single-base mismatched, two-base mismatched, three-base mismatched and four-base mismatched DNA of caspase-3, respectively. This is attributed to the optimized experimental conditions, with samples containing 2 μM of Mg(II) and 0.3 mM of dNTP. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Background: The aim of this project was to isolate and purify the highly active recombinant Taq DNA polymerase from the strain of Escherichia coli BL21. This enzyme, with a molecular weight of about 94 kDa, is widely used in polymerase chain reaction (PCR). In PCR, the activity and fidelity of Taq polymerase can significantly influence the results. One of the active regions of Taq polymerase has been suggested to be the O-helix region. In previous studies, an expression vector containing mutated Asn 666 Glu Taq polymerase gene was designed. In order to investigate the effects of this mutation on the function of the enzyme, Taq polymerase needs to be purified first. Methods: In this study, after transformation of competent cells, enzyme expression was induced by isopropyl â D thiogalactopyranoside (IPTG) method. Modified Desai protocol with DNase and RNase, nickel-nitrilotriacetic acid (Ni-NTA) resin, trichloroacetic acid (TCA), and refolding protocols were subsequently used for purification of this enzyme. These protocols were finally compared. Findings: Using Desai protocol resulted in the production of a sharp band in the expected region (94 kDa) and several other visible bands. After further modification of Desai protocol, only the desirable band was observed. In protocols using TCA and Ni-NTA resin, the expected bands were weak. Refolding protocol caused a band in an undesirable region (66 kDa). Conclusion: From the different purification techniques that were used in this study, the modified method of Desai containing RNase and DNase worked best. Addition of TCA can precipitate proteins that had not been affected by heat. Using Ni-NTA resin resulted in elimination of unwanted bands. However, the amount of Taq polymerase was also decreased. The extra band that was observed in refolding protocol was probably due to the presence of proteases that were isolated with inclusion body and could digest Taq polymerase. [ABSTRACT FROM AUTHOR]

DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, DNA labeling, mutagenesis, and other experiments. Thermostable DNA polymerases are especially useful and became quite valuable after the development of PCR technology. A DNA polymerase from Thermus aquaticus (Taq polymerase) is the most famous DNA polymerase as a PCR enzyme, and has been widely used all over the world. In this study, the gene fragments of the family A DNA polymerases were amplified by PCR from the DNAs from microorganisms within environmental soil samples, using a primer set for the two conserved regions. The corresponding region of the pol gene for Taq polymerase was substituted with the amplified gene fragments, and various chimeric DNA polymerases were prepared. Based on the properties of these chimeric enzymes and their sequences, two residues, E742 and A743, in Taq polymerase were found to be critical for its elongation ability. Taq polymerases with mutations at 742 and 743 actually showed higher DNA affinity and faster primer extension ability. These factors also affected the PCR performance of the DNA polymerase, and improved PCR results were observed with the mutant Taq polymerase. [ABSTRACT FROM AUTHOR]

Background: PCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical. Methodology/Principal Findings: This report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H2O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase. Conclusions/Significance: It is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved ''treatment-free'' attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample. [ABSTRACT FROM AUTHOR]

Formation of micro- and nanoparticles of condensed DNA during PCR with microbial genomic DNA or plasmid DNA as templates was reported previously. Initially, the microparticles were formed using a thermostable KlenTaq polymerase, which is a deletion variant of Taq polymerase. The present work shows that Taq polymerase is also capable of efficient formation of micro- and nanoparticles of condensed DNA in PCR. Electron microscopy revealed a number of morphological types (more than four) of microparticles produced in PCR with different reaction buffers in the presence of Taq polymerase and different plasmid DNAs as a template. In the case of some kinds of amplicons, an increase in the number of thermal cycles was shown to result in production of numerous nanowires and electron-dense spherical nanoparticles. The PCR conditions for preferential formation of discs (or ellipsoids) a few micrometers in diameter and several dozens of nanometers in thickness were determined. The structure of microparticles formed in the presence of Taq polymerase was found to depend on the level of synthesis of single-stranded DNA fragments in PCR. Experiments with nuclease S1 revealed that, along with double-stranded DNAs of the amplicon, micro- and nano-particles contained single-stranded DNA fragments, which were absolutely necessary for their formation. In light of these data, the molecular mechanism of micro- and nanoparticle formation in the course of PCR is discussed. [ABSTRACT FROM AUTHOR]

Standard Illumina libraries are biased toward sequences of intermediate GC-content. This results in an underrepresentation of GC-rich regions in sequencing projects of genomes with heterogeneous base composition, such as mammals and birds. We developed a simple, cost-effective protocol to enrich sheared genomic DNA in its GC-rich fraction by subtracting AT-rich DNA. This was achieved by heating DNA up to 90 °C before applying Illumina library preparation. We tested the new approach on chicken DNA and found that heated DNA increased average coverage in the GC-richest chromosomes by a factor up to six. Using a Taq polymerase supposedly appropriate for PCR amplification of GC-rich sequences had a much weaker effect. Our protocol should greatly facilitate sequencing and resequencing of the GC-richest regions of heterogeneous genomes, in combination with standard short-read and long-read technologies. [ABSTRACT FROM AUTHOR]

Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification. [ABSTRACT FROM AUTHOR]

Background: Accurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1–V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control. Results: Based on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92. Conclusions: These findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification. [ABSTRACT FROM AUTHOR]

Background: Accurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1–V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control. Results: Based on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92. Conclusions: These findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification. [ABSTRACT FROM AUTHOR]

We have developed a 9-week undergraduate laboratory series focused on the purification and characterization of Thermus aquaticus DNA polymerase (Taq). Our aim was to provide undergraduate biochemistry students with a full-semester continuing project simulating a research-like experience, while having each week's procedure focus on a single learning goal. The laboratory series has been taught for the past 7 years, and survey-based assessment of the effectiveness of the laboratory series was completed during the 2006 and 2007 fall semesters. Statistical analysis of the survey results demonstrate that the laboratory series is very effective in teaching students the theory and practice of protein purification and analysis while also demonstrating positive results in more broad areas of scientific skill and knowledge. Amongst the findings, the largest reported increases in knowledge were related to students' understanding of how patent law relates to laboratory science, a topic of great importance to modern researchers that is readily discussed in relation to Taq polymerase. Overall, this laboratory series proves to be a very effective component in the curricula of undergraduate biology and chemistry majors and may be an appropriate laboratory experience for undergraduates. [ABSTRACT FROM AUTHOR]

We propose a facile fluorometric system for detection of gene mutations using graphene oxide (GO). A fluorescent probe DNA anneals to a specific mutant gene and is degraded by the 5′→ 3′ exonuclease activity of Taq polymerase during PCR, and the released fluorophore retains fluorescence after addition of GO without quenching. [ABSTRACT FROM AUTHOR]

Taq polymerase system is one of the key success factors for genotyping by using semi-random marker types. Start codon targeted (SCoT) and sequence-related amplified polymorphism (SRAP) are semi-random markers that amplify more than one target at a time, like in multiplex PCR. Here, we compared the performance of five multiplex PCR kits (QIAGEN®, Thermo Scientific™, New England Biolabs, KAPA Biosystem and iNtRON Biotechnology) and Taq DNA polymerase from Vivantis as a representative common Taq polymerase. The PCR using three SCoT and three SRAP markers was performed in six plants, which were different in genome size, including three monocotyledons (rice, oil palm and onion) and three dicotyledons (holy basil, chilli pepper and eggplant). The overall results demonstrated consistent high performance of the Taq polymerase system with the multiplex PCR kit from KAPA Biosystem. Considered by plant, the multiplex PCR kit from KAPA Biosystem showed the best performance in holy basil, eggplant, oil palm and chilli pepper, whereas in rice, the Taq DNA polymerase from Vivantis generated better results than the KAPA Biosystem. In onion, multiplex PCR kit from Thermo Scientific™ showed the best performance among those tested, even if it did not perform well in the other plans. Different Taq polymerase systems might generate different band patterns, therefore using the same Taq polymerase system within one experimental dataset is recommended. The multiplex PCR kit from KAPA Biosystem is the best starting choice. [ABSTRACT FROM AUTHOR]

Ninety-nine randomly selected isolates of Listeria monocytogenes from several processing environment locations, in a shrimp processing plant, obtained during a 5-month sampling period were subjected to randomly amplified polymorphic DNA (RAPD) analysis with the use of four primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. Eighteen composite RAPD types were discerned with the use of the four primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates, and type 9 comprised 49 indistinguishable isolates. These results indicate that the shrimp processing plant was dominated by these 2 RAPD types that comprised 63.6% of the 99 randomly selected isolates. [ABSTRACT FROM AUTHOR]

Copyright of Baghdad Science Journal is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A simple and inexpensive method is reported for the long-term stabilization of enzymes and other unstable reagents in premeasured quantities in water-soluble tablets (cast, not compressed) made with pullulan, a nonionic polysaccharide that forms an oxygen impermeable solid upon drying. The pullulan tablets dissolve in aqueous solutions in seconds, thereby facilitating the easy execution of bioassays at remote sites with no need for special reagent handling and liquid pipetting. This approach is modular in nature, thus allowing the creation of individual tablets for enzymes and their substrates. Proof-of-principle demonstrations include a Taq polymerase tablet for DNA amplification through PCR and a pesticide assay kit consisting of separate tablets for acetylcholinesterase and its chromogenic substrate, indoxyl acetate, both of which are highly unstable. The encapsulated reagents remain stable at room temperature for months, thus enabling the room-temperature shipping and storage of bioassay components. [ABSTRACT FROM AUTHOR]

Ligictaluridus michaelalicea n. sp. (Monogenea: Dactylogyridae, Ancyrocephalinae) is described from the gills of Pylodictis olivaris (Siluriformes: Ictaluridae) from Wisconsin and Iowa portions of the upper Mississippi River. Diagnostic features include a relatively large, strongly curved tubular cirrus with minor terminal flare; an accessory piece with a prominent basal lobe and a simple, thick terminal limb featuring a thin lateral flange and blunt distal tip devoid of recurved hooks. The sinistral vagina, two prostatic reservoirs, and a terminal seminal vesicle of the vas deferens are prominent. The anchors, ventral and dorsal bars, and hooks are similar in overall form to those of other members of the genus. The description includes sequence data for the 18S rRNA gene, which aligned most closely with species of ancyrocephaline monogeneans, with the highest similarity being with Ligictaluridus pricei (Mueller, 1936). Other monogenean species identified from the flathead catfish examined included L. pricei and Ligictaluridus mirabilis (Mueller, 1937). L. michaelalicea n. sp. is the fourth species to be described from P. olivaris within its natural range in central and eastern North America. Implications resulting from taxonomic name changes, including species of Ligictaluridus, and United States Food and Drug Administration treatment regulations are discussed. An updated key to species of the genus Ligictaluridus is presented. [ABSTRACT FROM AUTHOR]

Several 7-(hydroxy, amino, methylureido, and guanidino)alkynyl-substituted 8-aza-7-deaza- hypoxanthine analogues were investigated as potential universal nucleobases. 7-Aminobutynyl-8-aza-7-deazahypoxanthine was found to be the most promising quasi-universal nucleobase with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used hypoxanthine (the nucleobase of inosine). It demonstrated improved ambiguity for pairing with A, T, and C bases and its base pairing properties can be summarized as follows: X:C∼X:A∼X:T > X:G. The improvement in PCR performance directly correlated with primer's Tm. Primers containing multiple 7-aminobutynyl-8-aza-7-deazahypoxanthines were successfully used without noticeable inhibition of Taq polymerase activity provided the modifications are positioned more than two bases away from the 3′ end. [ABSTRACT FROM PUBLISHER]

Polymerase chain reaction (PCR) is an in vitro technique to produce million fold copies of a particular segment of DNA. PCR should optimally yield a unique product unless designed otherwise. Artefacts appear many times, which affect the success of downstream applications. Prevention of non specific amplification using formamide and manipulation of cycling time is discussed in this article. [ABSTRACT FROM AUTHOR]

We present a new, general concept termed Hydrodynamic Thermal Confinement (HTC), and its implementation for the creation of microscale dynamic thermo-chemical microenvironments on biological surfaces. HTC is based on a scanning probe and operates under physiological conditions. The temperature can be regulated between 30° and 80 °C with ±0.2 °C precision and temperature ramps of 5 °C s−1 over a footprint of ∼50 μm × 80 μm in a volume of ∼50 × 80 × 15 μm3 (∼50 pl). [ABSTRACT FROM AUTHOR]

The use of molecular methods to investigate the community structure and diversity of microalgae has largely replaced the previous morphological methods that were routinely carried out by microscopy. Different DNA polymerases can lead to bias in PCR amplification and affect the downstream community and diversity analysis. In this study, two clone libraries were constructed with two different DNA polymerases, Q5 high-fidelity DNA polymerase and exTaq polymerase, to compare the differences in their capability to accurately reflect the cyanobacterial community structure and diversity in a subtropical deep freshwater reservoir, Dongzhen reservoir. The results indicated that the two cyanobacterial clone libraries constructed by using Q5 high-fidelity DNA polymerase and exTaq DNA polymerase did not show significant differences, although a slightly higher community diversity was revealed by using Q5 high-fidelity DNA polymerase. It is noteworthy that so far Q5 high-fidelity DNA polymerase was the first time to be employed in the genetic analysis of cyanobacterial community. And it is for the first time that the cyanobacterial community structure in Dongzhen reservoir was analyzed using molecular methods. Phylogenetic analysis revealed that most of the clones belonged to Cyanophyta and chloroplast, among which Cyanobium sp. Suigetsu-CR5 made up the largest fraction of cyanobacteria in winter. [ABSTRACT FROM AUTHOR]

Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve outbreak tracking and source apportionment. Here, we have developed such a method with a consensus-based bioinformatics pipeline and optimised reverse transcription (RT) and PCR procedures. Inhibitor removal and LunaScript® RT gave robust amplification of the ≈ 1000 bp RdRP + VP1 amplicon for both the GI and GII PCR assays. Platinum™ Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3 and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also frequently detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population. [ABSTRACT FROM AUTHOR]

Nucleic acid testing (NAT) is important for the identification and quantification of specific nucleic acid targets, both DNA and RNA, in life sciences and clinical diagnostics. Nucleic acid amplification can be a time-consuming step in NAT using the polymerase chain reaction (PCR) assay. Therefore, this study aimed to develop a simple method to reduce the amplification time while maintaining the PCR system. The three-step process of a general qPCR was reduced to a two-step process. The annealing/extension temperatures were increased to minimize the differences between the denaturation temperature and the annealing/extension temperatures. Subsequently, the time for each of these steps was reduced and, finally, the denaturation temperature was lowered. Taq polymerase was replaced with SD polymerase because it has strand displacement activity and is efficient in amplifying partial dsDNA at lower denaturation temperatures. In the two-step qPCR of genomic DNA using SD polymerase, the final conditions included an initial denaturation at 92 °C for 2 min, and 1 s at each cycling step with a denaturation temperature of 87 °C and an annealing/extension temperature of 72 °C. Amplification of the nucleocapsid (N) gene of SARS-CoV-2 RNA virus was evaluated at a template concentration as low as 10 copies. This method, named SF-qPCR (strand displacement-based fast quantitative polymerase chain reaction), can stably detect less than 10 copies of DNA and RNA within 25–40 min. This new protocol allows for sensitive and rapid detection of important DNA and RNA targets in clinical diagnosis. [ABSTRACT FROM AUTHOR]

Copyright of Vitae (01214004) is the property of Universidad de Antioquia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson-Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme. [ABSTRACT FROM AUTHOR]

Screening for oncogene mutations as a marker for malignancy can be a powerful tool for the early diagnosis of cancer. The enrichment polymerase chain reaction (PCR) is a sensitive method for the detection of low-frequency mutations in small samples. However, false-positive results, caused by methodological errors, may have severe clinical implications. When applied to the detection of Ki- ras mutations in pancreatic secretions, the assay sensitivity is limited to approximately 1:1400. Our investigation of Ki- ras mutations in blood samples from patients with pancreatic carcinoma revealed PCR bands presumably derived from mutant Ki- ras in samples from healthy volunteers, while all blood samples of the patients with pancreatic carcinomas showed a wild-type band pattern. Mathematical modeling of the PCR reaction reveals that the rate of false positive PCR results depends on the initial amount of DNA, the Taq polymerase error rate, the number of PCR reaction cycles, reaction efficiency and the restriction endonuclease chosen. The overall error rate of false positive results of the enrichment PCR can be reduced to the square of the rate of a single-step analysis if repeated amplifications of the same DNA specimen show an identical result. [ABSTRACT FROM AUTHOR]

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR. [ABSTRACT FROM AUTHOR]

Long-range conformational changes in proteins are ubiquitous in biology for the transmission and amplification of signals; such conformational changes can be triggered by small-amplitude, nanosecond protein domain motion. Understanding how conformational changes are initiated requires the characterization of protein domain motion on these timescales and on length scales comparable to protein dimensions. Using neutron spin-echo spectroscopy (NSE), normal mode analysis, and a statistical-mechanical framework, we reveal overdamped, coupled domain motion within DNA polymerase 1 from Thermus aquaticus (Taq polymerase). This protein utilizes correlated domain dynamics over 70 Å to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. We show that NSE spectroscopy can determine the domain mobility tensor, which determines the degree of dynamical coupling between domains. The mobility tensor defines the domain velocity response to a force applied to it or to another domain, just as the sails of a sailboat determine its velocity given the applied wind force. The NSE results provide insights into the nature of protein domain motion that are not appreciated by conventional biophysical techniques. [ABSTRACT FROM AUTHOR]

Here we develop a new method for the sensitive and selective detection of cell-surface proteins with an aptamer probe designed for binding-responsive catalysis of Taq DNA polymerase. Taking the biotin receptor as a model, the method allows the detection of target protein on surfaces of different types of cancer cells. [ABSTRACT FROM AUTHOR]

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity. D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer. [ABSTRACT FROM AUTHOR]

Background: The Cobas TaqMan MTB assay is used for rapid detection of the Mycobacterium tuberculosis complex (MTC) in clinical samples. It is only validated for respiratory samples, but is often requested by physicians for non-respiratory specimens. The aim of this study was therefore to evaluate the performance of this assay in clinical praxis in a country with low prevalence of tuberculosis (TB). Methods: The results from 2388 respiratory and 1005 non-respiratory human specimens were analyzed by this real-time PCR technique. Using culture results as the gold standard, the sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) of the PCR results were calculated. Results: For smear-positive respiratory specimens all the four values investigated were 100%. The number of smear-positive nonrespiratory specimens was only eight, and all of these eight specimens reacted positively. Sensitivity was 51% for smearnegative respiratory specimens, and 47% for smear-negative non-respiratory specimens. When all non-respiratory specimens were analyzed together the sensitivity was 51%. Specimens from 16 patients were PCR-positive but culture-negative and these cases are discussed. In one case, TB DNA was still present in sputum 2 years after a successful treatment. Conclusion: The study shows that the Cobas TaqMan MTB assay performs well in smear-positive samples, while the sensitivities are unsatisfactory for both respiratory and non-respiratory smear-negative specimens. Furthermore, the analyses emphasize that this assay cannot be used to evaluate treatment or contagiousness, or to detect relapses or screen for TB. [ABSTRACT FROM AUTHOR]

CYP2C19 is a polymorphic enzyme that metabolizes a wide variety of therapeutic drugs that has been associated with altered enzymatic activity and adverse drug reactions. Differences in allele frequencies of the CYP2C19 gene have been detected in populations worldwide. Thus, we analysed the alleles CYP2C19*2, CYP2C19*3, CYP2C19*4 and CYP2C19*5 related to the poor metabolizer (PM) phenotype in a Mexican population sample (n = 238), as well as CYP2C19*17, unique allele related to ultrarapid metabolizer phenotype (UMs). Genotypes were determined using SNaPshot and TaqManqPCR assays. In addition to the wild-type CYP2C19*1 allele (77.1%), we only found CYP2C19*17 (14.3%) and CYP2C19*2 (8.6%). Comparison with previous population reports demonstrated that these two SNPs are homogeneously distributed in Latin America (P > 0.05). Based on comparison with a previous pharmacokinetic study that determined the frequency of CYP2C19 phenotypes in the same population (western Mexican), we obtained the following findings: (i) based on the difference between the frequency of genotypes CYP2C19*2/*2 (presumably PM) versus the observed prevalence of PM phenotypes (0.4 versus 6.3%; X² = 9.58, P = 0.00196), we inferred the plausible presence of novel CYP2C19 alleles related to the PM phenotype; (ii) the prevalence of UMs was in disagreement with the dominant inheritance pattern suggested for CYP2C19*17 (23.1 versus 4%; P < 0.00001); (iii) the apparent recessive inheritance pattern of CYP2C19*17, based on the agreement between homozygous CYP2C19*17/*17 (presumably UMs) and the observed prevalence of UMs (2.1 versus 4%; (X² = 1.048; P = 0.306). [ABSTRACT FROM AUTHOR]

It has been suggested that Wigner's clock inequalities can be applied to biological systems. Pešsić was the first to suggest this by applying these inequalities to the reproduction time of a mycoplasma (1993 Eur. J. Phys. 14 90). More recently, Goel applied Wigner's clock inequalities to the system formed by a Taq DNA polymerase reading a phage lambda DNA strand, concluding that the behaviour of polymerases is governed by quantum mechanical processes. By means of a counterexample, using a Pfu DNA polymerase, I show that current experimental evidence does not allow us to conclude that Wigner's inequalities govern the behaviour of polymerases. Furthermore, both works are based on an incorrect interpretation of the concept of position uncertainty. [ABSTRACT FROM AUTHOR]

The red-spot prawn, Metapenaeopsis barbata, is a commercially important, widely distributed demersal species in the Indo-West Pacific Ocean. Overfishing has made its populations decline in the past decade. To study conservation genetics, eight polymorphic microsatellite loci were isolated. Genetic characteristics of the SSR (simple sequence repeat) fingerprints were estimated in 61 individuals from adjacent seas of Taiwan and China. The number of alleles, ranging from 2 to 4, as well as observed and expected heterozygosities in populations, ranging from 0.048 to 0.538, and 0.048 and 0.654, respectively, were detected. No deviation from Hardy-Weinberg expectations was detected at either locus. No significant linkage disequilibrium was detected in locus pairs. The polymorphic microsatellite loci will be useful for investigations of the genetic variation, population structure, and conservation genetics of this species. [ABSTRACT FROM AUTHOR]

Background: Over the last ten years we have seen great efforts focused on revising amphibian systematics. Phylogenetic reconstructions derived from DNA sequence data have played a central role in these revisionary studies but have typically under-sampled the diverse frog family Microhylidae. Here, we present a detailed phylogenetic study focused on expanding previous hypotheses of relationships within this cosmopolitan family. Specifically, we placed an emphasis on assessing relationships among New World genera and those taxa with uncertain phylogenetic affinities (i.e., incertae sedis). Results: One mitochondrial and three nuclear genes (about 2.8 kb) were sequenced to assess phylogenetic relationships. We utilized an unprecedented sampling of 200 microhylid taxa representing 91% of currently recognized subfamilies and 95% of New World genera. Our analyses do not fully resolve relationships among subfamilies supporting previous studies that have suggested a rapid early diversification of this clade. We observed a close relationship between Synapturanus and Otophryne of the subfamily Otophryninae. Within the subfamily Gastrophryninae relationships between genera were well resolved. Conclusion: Otophryninae is distantly related to all other New World microhylids that were recovered as a monophyletic group, Gastrophryninae. Within Gastrophryninae, five genera were recovered as non-monophyletic; we propose taxonomic re-arrangements to render all genera monophyletic. This hypothesis of relationships and updated classification for New World microhylids may serve as a guide to better understand the evolutionary history of this group that is apparently subject to convergent morphological evolution and chromosome reduction. Based on a divergence analysis calibrated with hypotheses from previous studies and fossil data, it appears that microhylid genera inhabiting the New World originated during a period of gradual cooling from the late Oligocene to mid Miocene. [ABSTRACT FROM AUTHOR]

Presents the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft. Structural diagrams; Finding that part of the DNA bound to the polymerase site shares a common binding site with DNA bound to the exonuclease site.