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Biochemistry. Biokhimiia Nov 2023Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage...
Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.
Topics: Humans; DNA-Directed DNA Polymerase; A549 Cells; Cisplatin; Hydrogen Peroxide; DNA Replication; DNA Damage; Adenocarcinoma of Lung; DNA Primase; Multifunctional Enzymes
PubMed: 38105210
DOI: 10.1134/S0006297923110214 -
Current Pharmaceutical Design 2024Digital PCR (dPCR) is the latest technique that has become commercially accessible for various types of research. This method uses Taq polymerase in a standard... (Review)
Review
Digital PCR (dPCR) is the latest technique that has become commercially accessible for various types of research. This method uses Taq polymerase in a standard polymerase chain reaction (PCR) to amplify a target DNA fragment from a complex sample, like quantitative PCR (qPCR) and droplet digital PCR (dd- PCR). ddPCR may facilitate microRNA (miRNA) measurement, particularly in liquid biopsy, because it has been proven to be more effective and sensitive, and in this method, ddPCR can provide an unprecedented chance for deoxyribonucleic acid (DNA) methylation research because of its capability to increase sensitivity and precision over conventional PCR-based methods. qPCR has also been found to be a valuable standard technique to measure both copy DNA (cDNA) and genomic DNA (gDNA) levels, although the finding data can be significantly variable and non-reproducible without relevant validation and verification of both primers and samples. The SYBR green quantitative real-time PCR (qPCR) method has been reported as an appropriate technique for quantitative detection and species discrimination, and has been applied profitably in different experiments to determine, quantify, and discriminate species. Although both TaqMan qRT-PCR and SYBR green qRT-PCR are sensitive and rapid, the SYBR green qRT-PCR assay is easy and the TaqMan qRT-PCR assay is specific but expensive due to the probe required. This review aimed to introduce dPCR, qPCR, SYBR green PCR kit, and digital PCR, compare them, and also introduce their advantages in the detection of different diseases.
Topics: Humans; Real-Time Polymerase Chain Reaction; Benzothiazoles; Diamines; DNA; Quinolines
PubMed: 38243947
DOI: 10.2174/0113816128276560231218090436 -
Veterinary World Aug 2023Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the...
BACKGROUND AND AIM
Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV).
MATERIALS AND METHODS
The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method.
RESULTS
The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 10 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV.
CONCLUSION
The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan.
PubMed: 37766711
DOI: 10.14202/vetworld.2023.1682-1689 -
Analytical Chemistry Nov 2023Nonspecific amplification is a serious issue in DNA detection as it can lead to false-positive results and reduce specificity. It is very important to well understand...
Nonspecific amplification is a serious issue in DNA detection as it can lead to false-positive results and reduce specificity. It is very important to well understand its mechanism through sequencing nonspecific products. Here, an approach is developed using a nanopore sequencing technique after acquiring the long repetitive sequence of DNA products from nonspecific amplification. Based on the sequencing results, a new mechanism of nonspecific amplification designated as dynamic mismatched primer binding (DMPB) with the background DNA (bgDNA) is proposed. Unexpectedly, our findings show that the primers (∼20 nt) can bind to bgDNA for primer extension when only 6-11 fully matched (9-14 mismatched) base pairs are formed. After the single-stranded DNAs (ssDNAs) attached to the first primer are produced, more interestingly, with the aid of DNA polymerase, the other primer can bind to these ssDNAs in the case that the fully matched base pairs formed between them are even shorter than 6 bp. As a result, perfect "seeds" for polymerase chain reaction with information on both primers are produced so that exponential nonspecific amplification can occur. The DMPB mechanism can explain nonspecific amplification in other approaches as well. Finally, a mini-hairpin DNA is used to effectively inhibit nonspecific amplification by preventing the formation of an unexpected primer-bgDNA complex.
Topics: DNA; Polymerase Chain Reaction; DNA Primers; DNA-Directed DNA Polymerase; Repetitive Sequences, Nucleic Acid; DNA, Single-Stranded; Nucleic Acid Amplification Techniques
PubMed: 37922263
DOI: 10.1021/acs.analchem.3c02274 -
Marine Biotechnology (New York, N.Y.) Apr 2024The starfish Asterias amurensis, a well-known predator of molluscan species in intertidal ecosystems, has caused substantial ecological and economic losses in North...
The starfish Asterias amurensis, a well-known predator of molluscan species in intertidal ecosystems, has caused substantial ecological and economic losses in North China such as offshore Qingdao. Effective monitoring and prevention measures are urged to minimize its negative impacts. Compared with traditional biomonitoring methods, environmental DNA technology has emerged as a powerful and cost-efficient tool for inferring species' presence and abundance. In this study, we developed a pair of species-specific primers (i.e., Ast-F and Ast-R) for the A. amurensis mitochondrial COI gene and tested its utility in amplifying and quantifying the DNA fragments from environmental samples under both laboratory and field conditions. The results of controlled water tank experiments demonstrated that the amount of eDNA released by A. amurensis was positively related to its biomass; after the removal of the starfish, the eDNA degraded significantly in 24 h and remained detectable for 8 days. The number of eDNA copies enriched tended to increase with smaller pore size of filter membrane and larger volume of filtered water. For field tests, we confirmed the validation of our approach in six locations in Qingdao by filtering 1000 ml water per sample with a 0.45-µm pore size filtration. All the amplification products generated a single and bright band via gel electrophoresis, and the quantitative PCR results unveiled significant differences in eDNA copies. This study provided an eDNA-based approach for investigating the distribution and biomass of A. amurensis, which may help to formulate early warning and management strategies in coastal Qingdao and other regions.
Topics: Animals; DNA, Environmental; Asterias; DNA Primers; Species Specificity; China; Environmental Monitoring; Electron Transport Complex IV; Polymerase Chain Reaction; Starfish; DNA, Mitochondrial
PubMed: 38341825
DOI: 10.1007/s10126-024-10292-1 -
Plant Disease Jul 2023Viticulture is a traditional branch of agriculture in the Czech Republic. Grapevines (Vitis vinifera L.) are cultivated on more than 18,000 hectares in the wine-growing...
Viticulture is a traditional branch of agriculture in the Czech Republic. Grapevines (Vitis vinifera L.) are cultivated on more than 18,000 hectares in the wine-growing regions of Bohemia and South Moravia. South Moravia alone accounts for more than 90 % of the total wine-growing area in the country. Grapevine yellows are a complex of diseases associated with the phytoplasma presence. Phytoplasmas of at least five different groups can cause similar symptoms in grapevines, and they can be distinguished only on a molecular basis (EPPO 2016). One of them, the grapevine Flavescence dorée phytoplasma (GFDP), which belongs to the 16SrV group, is listed in Annex II, Part B, of the Commission Implementing Regulation (EU) 2019/2072 of 28 November 2019 as a Union quarantine pest known to occur in the Union territory. Official surveys for GFDP in the Czech Republic have been carried out since 2007. In 2016, the first occurrence of Scaphoideus titanus Ball, 1932, the main vector of GFDP, was reported in the South Moravian Region (EPPO Reporting Service 2016). This is a matter of concern because it indicates that there is a risk of disease dissemination to other geographical locations. In September 2021, a total of 250 samples of V. vinifera (preferentially focused on symptomatic plants) and four samples of the wild plant host Clematis vitalba L. were collected from 50 vineyards in South Moravia. Total DNA was extracted using High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). For phytoplasma screening, a real-time PCR test for generic detection of phytoplasmas was used (Christensen et al. 2004). Samples evaluated as positive were further tested by PCR using phytoplasma universal P1 and P7 primers (Deng and Hiruki 1991; Schneider et al. 1995), followed by nested PCR using the 16SrV group-specific primers fB1 and rULWS1 (Smart et al. 1996). For identification of 16SrV phytoplasma, sequence analysis of the secY-map genetic locus was performed. Two sets of primers were used: FD9f5/MAPr1 primers for the first PCR and FD9f6/MAPr2 for the nested PCR (Arnaud et al. 2007) with PCRBIO TaqMix (PCR Biosystems, London, UK). The nested PCR products were purified and sequenced (Eurofins Genomics, Ebersberg, Germany). The sequences were compared with sequences from the GenBank database. Phytoplasma of the 16SrV group was detected in three samples: V. vinifera cv. Gewürztraminer with symptoms of leaf reddening with no rolling and no other typical symptoms; C. vitalba L. with leaf curling (Fig. 1A); symptomless C. vitalba. The obtained sequences of the secY-map locus of all three 16SrV-positive samples were identical to the sequence of GFDP, isolate Vv-SI257 (Acc. No. FN811141), detected in grapevine in Tuscany (Italy), which belongs to 16SrV group. The sequence of the V. vinifera cv. Gewürztraminer isolate was submitted to GenBank under Acc. No. OQ185203. This isolate belongs to the Map-FD3 cluster (Fig. 1B), and the genotype identified is M51 (corresponding to FD-C), which has already been found in C. vitalba and outbreaks of Flavescence dorée in grapevines in some other European countries (Malembic-Maher et al. 2020). Based on the abovementioned results, this is the first report of the GFDP in the Czech Republic.
PubMed: 37467129
DOI: 10.1094/PDIS-02-23-0315-PDN -
Advanced Materials (Deerfield Beach,... Apr 2024Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for...
Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL. Compared to a quantitative real-time polymerase chain reaction assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.
PubMed: 38657970
DOI: 10.1002/adma.202311634 -
BMC Plant Biology Oct 2023The aim of this study was to evaluate the impact of salt stress on morphological, yield, biochemical, and molecular attributes of different barley genotypes. Ten...
The aim of this study was to evaluate the impact of salt stress on morphological, yield, biochemical, and molecular attributes of different barley genotypes. Ten genotypes were cultivated at Fayoum Research Station, El-Fayoum Governorate, Egypt, during two seasons (2020-2021 and 2021-2022), and they were exposed to two different salt concentrations (tap water as a control and 8000 ppm). The results showed that genotypes and salt stress had a significant impact on all morphological and physiological parameters. The morphological parameters (plant height) and yield attributes (spike length, number of grains per spike, and grain yield per plant) of all barley genotypes were significantly decreased under salt stress as compared to control plants. Under salt stress, the total soluble sugars, proline, total phenol, total flavonoid, ascorbic acid, malondialdehyde, hydrogen peroxide, and sodium contents of the shoots of all barley genotypes significantly increased while the potassium content decreased. L1, which is considered a sensitive genotype was more affected by salinity stress than the tolerance genotypes L4, L6, L9, and Giza 138. SDS-PAGE of seed proteins demonstrated high levels of genetic variety with a polymorphism rate of 42.11%. All genotypes evaluated revealed significant variations in the seed protein biochemical markers, with new protein bands appearing and other protein bands disappearing in the protein patterns of genotypes cultivated under various conditions. Two molecular marker techniques (SCoT and ISSR primers) were used in this study. Ten Start Codon Targeted (SCoT) primers exhibited a total of 94 fragments with sizes ranging from 1800 base pairs to 100 base pairs; 29 of them were monomorphic, and 65 bands, with a polymorphism of 62.18%, were polymorphic. These bands contained 21 unique bands (9 positive specific markers and 12 negative specific markers). A total of 54 amplified bands with molecular sizes ranging from 2200 to 200 bp were produced using seven Inter Simple Sequence Repeat (ISSR) primers; 31 of them were monomorphic bands and 23 polymorphic bands had a 40.9% polymorphism. The techniques identified molecular genetic markers associated with salt tolerance in barley crop and successfully marked each genotype with distinct bands. The ten genotypes were sorted into two main groups by the unweighted pair group method of arithmetic averages (UPGMA) cluster analysis based on molecular markers and data at a genetic similarity coefficient level of 0.71.
Topics: Hordeum; Genetic Variation; Genotype; Genetic Markers; DNA Primers; Salt Tolerance
PubMed: 37899447
DOI: 10.1186/s12870-023-04550-y -
Scientific Reports May 2024Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of...
Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.
Topics: SARS-CoV-2; Software; Polymerase Chain Reaction; Oligonucleotides; COVID-19; Computational Biology; DNA, Single-Stranded
PubMed: 38750104
DOI: 10.1038/s41598-024-59497-3 -
Parasites & Vectors Aug 2023Six species of apicomplexan parasites of the genus Babesia, namely B. microti, B. divergens, B. duncani, B. motasi, B. crassa-like and B. venatorum, are considered to be...
BACKGROUND
Six species of apicomplexan parasites of the genus Babesia, namely B. microti, B. divergens, B. duncani, B. motasi, B. crassa-like and B. venatorum, are considered to be the primary causal agents of human babesiosis in endemic areas. These six species possess variable degrees of virulence for their primary hosts. Therefore, the accurate identification of these species is critical for the adoption of appropriate therapeutic strategies.
METHODS
We developed a real-time PCR-high-resolution melting (qPCR-HRM) approach targeting 18S ribosomal RNA gene of five Babesia spp. based on melting temperature (T) and genotype confidence percentage values. This approach was then evaluated using 429 blood samples collected from patients with a history of tick bites, 120 DNA samples mixed with plasmids and 80 laboratory-infected animal samples.
RESULTS
The sensitivity and specificity of the proposed qPCR-HRM method were 95% and 100%, respectively, and the detection limit was 1-100 copies of the plasmid with the cloned target gene. The detection level depended on the species of Babesia analyzed. The primers designed in this study ensured not only the high interspecific specificity of our proposed method but also a high versatility for different isolates from the same species worldwide. Additionally, the Tm obtained from the prepared plasmid standard is theoretically suitable for identifying isolates of all known sequences of the five Babesia species.
CONCLUSIONS
The developed detection method provides a useful tool for the epidemiological investigation of human babesiosis and pre-transfusion screening.
Topics: Animals; Humans; Babesia; Babesiosis; Cloning, Molecular; DNA Primers; Gastropoda
PubMed: 37641091
DOI: 10.1186/s13071-023-05839-5