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Detection of genome editing with quantitative polymerase chain reaction (PCR) primarily relies on and is limited by its ability to discriminate genome modification from the wild-type sequence. An enhanced DNA polymerase variant with superior specificity is needed for this application. Here, we perform semi-rational molecular evolution on full-length Taq polymerase to screen high-specific variants that meet the requirements of gene variation detection. We substituted each of the 40 polar amino acids in direct contact with the primer/template duplex and conducted extensive random mutagenesis to generate a Taq mutation library. Screening on a quantitative PCR system with insertion and deletion-containing templates identified a series of improved Taq variants. We demonstrate that the Taq388 variant bearing three amino acid substitutions, S577A, W645R, and I707V, has improved sensitivity to insertion and deletion-derived primer/template mismatch by a ΔCt value of 25-26 and is superior for application in evaluating CRISPR-Cas9 editing efficiency and single-cell clone genotyping. In addition, the Taq variant shows substantial potential for single-nucleotide polymorphism detection by means of allele-specific PCR because of its high sensitivity to mismatches.

Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of

Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.

Polymerase chain reaction (PCR) is one of the most important techniques in molecular pathology by which the single or the pieces of target DNA are amplified by using a pair of DNA primer, heat-resistant DNA polymerase enzyme and nucleotides. This chapter discusses the principle, steps and application of PCR in pathology. There are four basic steps of PCR: denaturation, annealing and extension. The PCR thermal cycle rapidly heats and cools the PCR reagent mixture. The cycling time depends on (1) size of the DNA template and (2) G-C content of DNA. The number of the thermal cycler is usually set as 25–30 cycles. The PCR products are demonstrated by agarose gel electrophoresis of the product, cloning or sequencing of the products. The chapter also covers the troubleshooting of PCR. There are different types of PCR methods for diagnostic purposes that include reverse transcriptase PCR, asymmetric PCR, hot start PCR, in situ PCR, inverse PCR, single-strand conformation polymorphism, real-time PCR and nested PCR. All these types of PCR have been described in details.

The linkage between the occurrence of human leucocyte antigen B*27 (HLA-B*27) and ankylosing spondylitis or other related spondyloarthritides is well documented. PCR based methods are widely used for HLA-B*27 screening. To refine HLA-B*27 testing we aimed at establishing a real-time PCR protocol to detect the HLA-B*27 allele directly in blood samples, without DNA extraction. HLA-B*27 analysis was performed by two real-time PCRs using TaqMan primer-probe assays for B*27 specific amplification of exon 2 or exon 3 of the HLA-B gene together with a mutant of Taq polymerase for direct blood PCR. Conditions for direct blood PCR were optimized and the reliability of the direct blood PCR protocol was evaluated by re-genotyping over 200 blood samples from patients who previously underwent routine DNA-based HLA-B*27 testing. Heating blood samples at 95°C for 10 minutes significantly improved PCR performance. Results from real-time PCR based HLA-B*27 testing directly in blood of over 200 patients were in 100% concordance with results obtained by routine DNA-based HLA-B*27 genotyping. In summary, we present a reliable real-time PCR protocol for HLA-B*27 screening directly in whole blood supporting fast clarification of the presence of ankylosing spondylitis or other spondyloarthritides in suspected cases.

Classical restriction fragment length polymorphism (RFLP) and sequencing are labor-intensive and expensive methods to study single base changes, whereas polymerase chain reaction amplification of specific alleles (PASA) or allele-specific polymerase chain reaction (ASPCR) is a PCR-based application that allows direct detection of any point mutation by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. PASA is based on oligonucleotide primers containing one or more 3' mismatch with the target DNA making it refractory to primer extension by Thermus aquaticus DNA polymerase lacking the 3' to 5' exonuclease proofreading activity because of which it is also called amplification refractory mutation system-PCR (ARMS-PCR). This technique has found application in detection of allele, mutation, single-nucleotide polymorphisms (SNPs) causing genetic and infectious diseases. This chapter describes an approach of cohort PASA in context of genotyping single and double mutant worms generated to study the process of cell migration and axon outgrowth in C. elegans. Single worm-based cohort PASA allows genotyping for identification of single base mutations; particularly it is convenient method to detect mutations without a visible phenotype.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses’ genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV’s attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.

DNA modifying enzymes are ubiquitous reagents in synthetic biology. Producing these enzymes often requires large culture volumes, purified nucleases and chromatographic separations to make enzymes of necessary quality. We sought to leverage synthetic biology tools to develop engineered strains allowing for not only the production but rapid purification of these reagents. Toward this goal, we report an E. coli strain enabling the rapid production and purification of Taq polymerase. The method relies on 1) autoinducible expression achieving high protein titers, 2) autolysis and auto DNA/RNA hydrolysis via lysozyme and a mutant benzonase™, and 3) heat denaturation under reducing conditions to precipitate contaminating proteins including the mutant benzonase™. Taq polymerase is obtained at high purities (>95% pure by SDS-PAGE) and is readily usable in standard reactions. The method takes less than 1 hour of hands-on time, does not require special equipment, expensive reagents or affinity purification. We expect this simple methodology and approach will improve access not only to Taq polymerase but to numerous additional commonly utilized reagent proteins.HighlightsProtein titers ~ 1g/L achieved in 20 mL shakeflasks.4 mg of purified Taq (corresponding to 5,000 Units, or 4,000 PCR reactions) per 20 mL shake flask.Instant Taq is equivalent to commercial preparations in routine PCR

Rapid and sensitive detection of human pathogens, such as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is an urgent and challenging task for clinical laboratories. Currently, the gold standard test for SARS-CoV-2–specific RNA is based on quantitative RT-PCR (RT-qPCR), which relies on target amplification by Taq polymerase and uses a fluorescent resonance energy transfer–based hydrolysis probe. Although this method is accurate and specific, it is also time consuming. To rapidly detect the presence of the viral RNA in clinical samples, we describe a new molecular assay that combines a highly sensitive magnetic modulation biosensing (MMB) system, rapid thermal cycling, and a modified double-quenched hydrolysis probe. Using in vitro transcribed SARS-CoV-2 RNA targets spiked in PCR-grade water, we found that the calculated limit of detection of the MMB-based molecular assay was 1.6 copies per reaction. Testing 309 RNA extracts from 170 confirmed RT-qPCR SARS-CoV-2–negative individuals (30 of whom were positive to other respiratory viruses) and 139 RT-qPCR SARS-CoV-2–positive patients (CT ≤ 42) resulted in 97.8% sensitivity, 100% specificity, and 0% cross-reactivity. The total turnaround time of the MMB-based assay is 30 minutes, which is three to four times faster than a standard RT-qPCR. By adjusting the primers and the probe set, the platform can be easily adapted to detect most of the pathogens that are currently being diagnosed by RT-qPCR.

Simple Summary Minimizing silage additives cost while increasing silage quality is important for a sustainable livestock enterprise, especially in undeveloped and developing countries. In this study, therefore, commercially available kefir yeast (CK) and homemade kefir culture (HK), as a low-cost additive, was applied at untreated a common control (CON) and three different application doses (5.0, 5.7, and 6.0 log cfu g−1) on wilted alfalfa and evaluated with the fermentation characteristics and aerobic stability. The addition of HK with an application dose greater than 5.0 log cfu g−1 prevents mold formation and inhibits yeast counts in silages. Indeed, both CK and HK improve the silage quality and aerobic stability of alfalfa even with low water-soluble carbohydrate content. Abstract The present study has been one of the first attempts to thoroughly examine the effects of different kefir sources on fermentation characteristics, aerobic stability, and microbial communities of alfalfa silages. The effects of commercial kefir (CK) and homemade kefir culture (HK) applied with untreated a common control (CON) and three different application doses (5.0, 5.7, and 6.0 log cfu g−1) on wilted alfalfa and stored at an ambient temperature of 25–30 °C are studied. After 45 days of ensiling, fermentation characteristics and aerobic stability of silages were measured, and bacterial diversity was investigated by 16S ribosomal RNA gene sequencing using the GenomeLab™ GeXP platform. Both CK and HK accelerate more lactic acid production and reduced ammonia nitrogen concentration. Factor analysis of kefir sources suggests that the addition of kefir improves the aerobic stability of silages, even the initial water-soluble carbohydrate (WSC) content is inadequate via its antimicrobial effect on yeast and mold formation. Enterococcus faecium, Pediococcus pentosaceous and Lactobacillus brevis were dominant bacterial species among the treated groups at silo opening, while Lactobacillus plantarum and Lactobacillus brevis became dominant bacterial species after 7 days of aerobic exposure. In conclusion, the application of kefir on alfalfa silages improves fermentation quality and aerobic stability even with low WSC content.

As a powerful tool for obtaining sufficient DNA from rare DNA resources, polymerase chain reaction (PCR) has been widely used in various fields, and the optimization of PCR is still in progress due to the dissatisfactory specificity, sensitivity and efficiency. Although many nanomaterials have been proven to be capable of optimizing PCR, their underlying mechanisms are still unclear. So far, the scientifically compelling and functionally evolving metal–organic framework (MOF) materials with high specific surface area, tunable pore sizes, alterable surface charges and favourable thermal conductivity have not been used for PCR optimization. In this study, UiO-66 and ZIF-8 were used to optimize error-prone two round PCR. The results demonstrated that UiO-66 and ZIF-8 not only enhanced the sensitivity and efficiency of the first round PCR, but also increased the specificity and efficiency of the second round PCR. Moreover, they could widen the annealing temperature range of the second round PCR. The interaction of DNA and Taq polymerase with MOFs may be the main reason. This work provided a candidate enhancer for PCR, deepened our understanding on the enhancement mechanisms of nano-PCR, and explored a new application field for MOFs., Many new materials have the ability to optimize polymerase chain reaction (PCR). Metal-organic frame materials UiO-66 and ZIF-8 can enhance sensitivity, specificity and efficiency of PCR, indicating their potential as PCR enhancers.

During polymerase chain reaction (PCR), DNA polymerases such as Taq add a single, unpaired residue—preferentially an adenosyl residue—to each 3′ end of a double-stranded amplified product. The unpaired terminal (A) residues can pair with a linear T vector that carries an unpaired 3′-thymidyl residue at each end. T vectors can be prepared in the laboratory or they may be purchased ready-made from commercial suppliers. This protocol outlines two methods in common use to generate T vectors.

Cell free DNA (cfDNA) in human plasma carries abundant information, which has therefore been the key sample for noninvasive prenatal testing (NIPT) and liquid biopsy. Especially by using the rapidly developed next-generation sequencing (NGS) techniques, the genetic and epigenetic information embedded in cfDNA has been effectively and extensively decoded. In this process, a key step is to construct the NGS library. Due to its high degradation, the single strand-based method was reported to be more qualified to construct the NGS library of cfDNA. In order to develop a new simple method for this application, this study adapted our recently developed single strand adaptor library preparation (SALP) method for constructing an NGS library of cfDNA. In the improved method, cfDNA was firstly denatured into single strands and then ligated with a single strand adaptor (SSA) that had a 3' overhang of 3 random bases by using T4 DNA ligase. The SSA-ligated DNA was converted into double-stranded DNA with an additional adenine at the other end by polymerizing with Taq polymerase. Next, a barcode T adaptor (BTA) was ligated to this end. Finally, the cfDNA ligated with two adaptors was amplified with the Illumina-compatible primers for NGS. Using the method, this study successfully sequenced 20 cfDNA samples from 16 esophageal cancer patients and 4 healthy people. By bioinformatics analysis, this study found the genetic and epigenetic difference between patients and healthy people and identified 23 epigenetic and 28 genetic altered esophageal cancer-specific genes.

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as ‘negative’ under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene’s proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5–2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8–2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).

The substitution-inert polynuclear platinum complexes (SI-PPCs) are now recognized as a distinct subclass of platinum anticancer drugs with high DNA binding affinity. Here, we investigate the effects of SI-PPCs containing dangling amine groups in place of NH3 as ligands to increase the length of the molecule and therefore overall charge and its distribution. The results obtained with the aid of biophysical techniques, such as total intensity light scattering, gel electrophoresis, and atomic force microscopy, show that addition of dangling amine groups considerably augments the ability of SI-PPCs to condense/aggregate nucleic acids. Moreover, this enhanced capability of SI-PPCs correlates with their heightened efficiency to inhibit DNA-related enzymatic activities, such as those connected with DNA transcription, catalysis of DNA relaxation by DNA topoisomerase I, and DNA synthesis catalyzed by Taq DNA polymerase. Thus, the addition of the dangling amine groups resulting in structures of SI-PPCs, which differ so markedly from the derivatives of cisplatin used in the clinic, appears to contribute to the overall biological activity of these molecules.

In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with blaTEM gene fragments that varied in length and quantities. The complete blaTEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating blaTEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the blaTEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.

Context: Staphylococcus aureus is a Gram positive bacterium which has an impending ability to cause an array of infection ranging from skin lesions to life threatening systemic illness. In the past few decades there is a drastic rise in Methicillin resistant Staphylococcus aureus (MRSA). The acquisition of Staphylococcal cassette chromosome mec (SCC mec) carrying mecA gene leads to Methicillin resistance among Staphylococcus aureus. Aim: The aim of this study is to identify the prevalence of MRSA and identify the SCC mecA and Panton-Valentine Leukocidin (PVL) gene by Polymerase chain reaction (PCR) among Staphylococcus aureus among various clinical isolates in our hospital. Materials and Methods: A prospective cross-sectional study was conducted in a tertiary care hospital at Puducherry. The study was conducted over a period of 1year from January 2014 to December 2014. The isolates were obtained from various clinical samples such as pus, aspirate, urine, blood and sputum. PCR amplification of mecA and pvl genes was performed to check their prevalence among the isolates collected from a tertiary care hospital. PCR reaction mixtures (10?l) contained 1?lTaq PCR buffer (10X) with 15mM MgCl2, 0.1 µl (0.5U) of Taq polymerase (New England Biolabs, US), 0.25mM of dNTP mix (Thermo Scientific, US), 0.25pM each of forward and reverse primers (Eurofins, Bangalore, India) and 10ng of genomic DNA template. Results: Around 228 samples of Staphylococcus aureus isolated from various clinical samples were analyzed for methicillin sensitivity. Out of these samples around 49 (21.49%) samples were MRSA positive isolates. Out of 49 mecA harboring isolates pvl gene was amplified in 26 (53%) isolates. Conclusion: MRSA being a dreadful pathogen has minimal treatment options. PVL gene with added virulence further worsens the clinical outcome among infected patients. Hence the knowledge of its prevalence adds an insight among the infection control practitioners to adhere effective preven

DNA polymerase I from Thermus aquaticus ( Taq DNA polymerase) is useful for polymerase chain reactions because of its exceptional thermostability; however, its activity at low temperatures can cause amplification of unintended products. Mutation of isoleucine 707 to leucine (I707L) slows Taq DNA polymerase at low temperatures, which decreases unwanted amplification due to mispriming. In this work, unrestrained molecular dynamics (MD) simulations were performed on I707L and wild-type (WT) Taq DNA polymerase at 341 and 298 K to determine how the mutation affects the dynamic nature of the protein. The results suggest that I707L Taq DNA polymerase remains relatively immobile at room temperature and becomes more flexible at the higher temperature, while the WT Taq DNA polymerase demonstrates less substantial differences in dynamics at high and low temperatures. These results are in agreement with previous experimental results on the I707L mutant Taq DNA polymerase that show dynamic differences at high and low temperatures. The decreased mobility of the mutant at low temperature suggests that the mutant remains longer in the blocked conformation, and this may lead to reduced activity relative to the WT at 298 K. Principal component analysis revealed that the mutation results in decoupled movements of the Q helix and fingers domain. This decoupled nature of the mutant gives way to an increasingly flexible N-terminal end of the Q helix at 341 K, a characteristic not seen for WT Taq DNA polymerase.

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT‐qPCR) analysis. Commercial one‐step master mixes—which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well—are key reagents for SARS‐CoV‐2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M‐MLV reverse transcriptase and assemble a homemade one‐step RT‐qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS‐CoV‐2 RNA detection by RT‐qPCR: heat‐inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT‐qPCR using the homemade master mix, how to prepare in vitro−transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT‐PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one‐step RT‐qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT‐PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One‐step RT‐qPCR of RNA samples using a real‐time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One‐step RT‐PCR using endpoint fluorescence detection

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

Aims F11R gene encodes junctional adhesion molecule-A protein (JAM-A), which is expressed in various types of cells and is involved in leukocyte extravasation during inflammation. Sjogren's syndrome (SS) is a chronic systemic inflammatory disease that involves lymphocytes invasion of exocrine glands. F11R has been studied in autoimmune diseases, but any association between F11R and SS has not yet been investigated. Therefore, experiments were undertaken to examine the relationships among F11R gene polymorphism, messenger RNA (mRNA) expression and SS patients. Methods Three hundred and twenty-nine patients with SS, and 223 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. Genomic DNA was extracted from peripheral blood mononuclear cells and gene polymorphisms were genotyped by TaqMan real-time polymerase chain reaction (PCR). F11R mRNA expression was quantitated by quantitative real-time PCR with TaqMan Gene Expression Assay. Results Our study showed the genotype -688A/C (rs6695707) was not found in relation to SS patients. The odds ratio of -436A/G (rs12567886) genotype was notably associated with less susceptibility of SS in human leukocyte antigen (HLA)-DR2 negative and HLA-DR3 negative individuals. F11R mRNA expression was lower in SS patients than in the cells of healthy controls. Conclusion The result indicated that G allele of -436A/G genotype has the potential protective effect against SS disease condition. F11R mRNA was expressed significantly lower in SS patients.

Several native and engineered heat‐stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72–74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3–5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half‐life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high‐temperature DNA polymerization., The DNA polymerase from the extreme thermophile Thermus scotoductus strain K1 (TsK1) was expressed in Escherichia coli, purified, and characterized. The enzyme demonstrated an optimal temperature and pH value of 72–74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3–5 mM for optimal activity. The base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase.

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.

Polymerase chain reaction (PCR) is one of the most important techniques for prenatal diagnosis. It can amplify a specific DNA sequence from tiny fetal samples with high efficiency within a few hours. PCR is a cyclic DNA synthesis reaction by DNA polymerase in an automated system that amplifies the target sequence to over 100 million copies in a test tube. This technology is useful not only for invasive prenatal genetic diagnostic testing but also for noninvasive prenatal genetic testing.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase, and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.

Background: Amyloid-β peptide (Aβ) deposition in Alzheimer’s disease (AD) is due to an imbalance in its production/clearance rate. Aβ is transported across the blood-brain barrier by LRP1 and P-gp as efflux transporters and RAGE as influx transporter. Vitamin D deficit and polymorphisms of the vitamin D receptor (VDR) gene are associated with high prevalence of mild cognitive impairment (MCI) and AD. Further, vitamin D promotes the expression of LRP1 and P-gp in AD-animal model brains. Objective: To associate VDR polymorphisms Apa I (rs7975232), Taq I (rs731236), and Fok I (rs2228570) with the risk of developing MCI in a Chilean population, and to evaluate the relationship of these polymorphisms to the expression of VDR and Aβ-transporters in peripheral blood mononuclear cells (PBMCs). Methods: VDR polymorphisms Apa I, Taq I, and Fok I were determined in 128 healthy controls (HC) and 66 MCI patients. mRNA levels of VDR and Aβ-transporters were evaluated in subgroups by qPCR. Results: Alleles A of Apa I and C of Taq I were associated with a lower risk of MCI. HC with the Apa I AA genotype had higher mRNA levels of P-gp and LRP1, while the expression of VDR and RAGE were higher in MCI patients and HC. For Fok I, the TC genotype was associated with lower expression levels of Aβ-transporters in both groups. Conclusion: We propose that the response to vitamin D treatment will depend on VDR polymorphisms, being more efficient in carriers of protective alleles of Apa I polymorphism.