Enhancing APE1 detection through apurinic/apyrimidinic site inhibition of DNA polymerase: an innovative, highly sensitive approach.

This study presents an innovative method for the highly sensitive detection of apurinic/apyrimidinic endonuclease 1 (APE1), a crucial biomarker and target for cancer diagnosis and treatment. The method is predicated on our discovery that the apurinic or apyrimidinic site (AP site) can inhibit the activity of Taq DNA polymerase. Subsequent experiments further led to the development of a new amplification method based on the digestion activity of Lambda exonuclease. This approach showed potential to detect trace amounts of APE1 in biological samples with high sensitivity.

A Fusion of Taq DNA Polymerase with the CL7 Protein from Escherichia coli Remarkably Improves DNA Amplification.

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase.

AIMS: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of S. pneumoniae, N. meningitidis, H. influenzae, and serogroups and serotypes of these three pathogens. METHODS: Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation. RESULTS: All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3-4.7 and 1.2-3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase. CONCLUSIONS: It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of S. pneumoniae, N. meningitidis and H. influenzae at concentrations close to the lower limit of medical decision point.

Investigation of the stability of D5SIC-DNAM-incorporated DNA duplex in Taq polymerase binary system: a systematic classical MD approach.

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.

Optimized bacterial community characterization through full-length 16S rRNA gene sequencing utilizing MinION nanopore technology.

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.

Clamping-mediated incorporation of single-stranded DNA with concomitant DNA synthesis by Taq polymerase involves nick-translation.

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.

Development of a Simple Direct and Hot-Start PCR Using Escherichia coli-Expressing Taq DNA Polymerase.

Taq DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing Taq DNA polymerase for routine use in laboratories. We developed a method using Escherichia coli (E. coli) that expresses thermostable Taq DNA polymerase directly in the PCR without purification. The Taq gene was transformed into E. coli and expressed. After overnight incubation and washing, E. coli-expressing Taq DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.

Incorporation of N7-Platinated Guanines into Thermus Aquaticus (Taq) DNA Polymerase: Atomistic Insights from Molecular Dynamics Simulations.

In this work, we elucidated some key aspects of the mechanism of action of the cisplatin anticancer drug, cis-[Pt(NH3)2Cl2], involving direct interactions with free nucleotides. A comprehensive in silico molecular modeling analysis was conducted to compare the interactions of Thermus aquaticus (Taq) DNA polymerase with three distinct N7-platinated deoxyguanosine triphosphates: [Pt(dien)(N7-dGTP)] (1), cis-[Pt(NH3)2Cl(N7-dGTP)] (2), and cis-[Pt(NH3)2(H2O)(N7-dGTP)] (3) {dien = diethylenetriamine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate}, using canonical dGTP as a reference, in the presence of DNA. The goal was to elucidate the binding site interactions between Taq DNA polymerase and the tested nucleotide derivatives, providing valuable atomistic insights. Unbiased molecular dynamics simulations (200 ns for each complex) with explicit water molecules were performed on the four ternary complexes, yielding significant findings that contribute to a better understanding of experimental results. The molecular modeling highlighted the crucial role of a specific α-helix (O-helix) within the fingers subdomain, which facilitates the proper geometry for functional contacts between the incoming nucleotide and the DNA template needed for incorporation into the polymerase. The analysis revealed that complex 1 exhibits a much lower affinity for Taq DNA polymerase than complexes 2-3. The affinities of cisplatin metabolites 2-3 for Taq DNA polymerase were found to be quite similar to those of natural dGTP, resulting in a lower incorporation rate for complex 1 compared to complexes 2-3. These findings could have significant implications for the cisplatin mechanism of action, as the high intracellular availability of free nucleobases might promote the competitive incorporation of platinated nucleotides over direct cisplatin attachment to DNA. The study's insights into the incorporation of platinated nucleotides into the Taq DNA polymerase active site suggest that the role of platinated nucleotides in the cisplatin mechanism of action may have been previously underestimated.

Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

The Effect of γ Phosphate Modified Deoxynucleotide Substrates on PCR Activity and Fidelity.

Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A : T→G : C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G : C→A : T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

A Brief Practical Guide to PCR.

Polymerase chain reaction (PCR) has been a powerful molecular biology tool since the mid-1980s. Millions of copies of specific sequence regions of DNA can be generated to allow the study of these regions. Fields that use this technology range from forensics to the experimental study of human biology. Standards for performing PCR and information tools to help design PCR protocols aid in successful implementation of PCR.

[Comparative Analysis of Family A DNA-Polymerases as a Searching Tool for Enzymes with New Properties].

DNA polymerases catalyze DNA synthesis during DNA replication, repair, and recombination. A number of DNA polymerases, such as the Taq enzyme from Thermus aquaticus, are used in various applications of molecular biology and biotechnology, in particular as DNA amplification tools. However, the efficiency of these enzymes depends on factors such as DNA origin, primer composition, template length, GC-content, and the ability to form stable secondary structures. These limitations in the use of currently known DNA polymerases lead to the search for new enzymes with improved properties. This review summarizes the main structural and molecular-kinetic features of the functioning of DNA-polymerases belonging to structural family A, including Taq polymerase. A phylogenetic analysis of these enzymes was carried out, which made it possible to establish a highly conserved consensus sequence containing 62 amino acid residues distributed over the structure of the enzyme. A comparative analysis of these amino acid residues among poorly studied DNA-polymerases revealed 7 enzymes that potentially have the properties necessary for use in DNA amplification.

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences.

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5'-3' exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of GoAstV, GPV, and GoCV.

Goose astrovirus (GoAstV), goose parvovirus (GPV), and goose circovirus (GoCV) infections have similar symptoms, such as severe diarrhea, and cause serious economic losses to the goose industry globally. Therefore, it is necessary to develop a rapid and accurate method for the differential diagnosis of the 3 viruses. In this study, a TaqMan probe-based multiplex reverse transcription-qualitative polymerase chain reaction (RT-qPCR) method was established and optimized for simultaneous detection of the three viruses. Three pairs of specific primers and probes were designed considering the conserved sequences of ORF2, VP3, and Rep of GoAstV, GPV, and GoCV, respectively. Singleplex real-time RT-qPCR detected a minimum of 10 copies of these genes, while multiplex real-time RT-qPCR detected a minimum of 100 copies. The correlation coefficients exceeded 0.99, and the amplification efficiency was 80 to 100%. The assay had high sensitivity, specificity, and repeatability. In 85 tissue samples, GoAstV and GPV were the main pathogens and demonstrated co-infection. This assay provides a rapid, efficient, specific, and sensitive tool for the detection of GoAstV, GPV, and GoCV. This can facilitate disease management and epidemiological surveillance.

Low-level point heteroplasmy detection in human mitogenomes amplified with different polymerases and sequenced on MiSeq FGx platform.

Introduction: Massively parallel sequencing of mitogenomes usually requires prior amplification. The PCR step may influence the quality of the data obtained, especially when low-level heteroplasmy detection is applied. Aim: The aim of this study was to compare the reliability of two different DNA polymerases in detecting homoplasmic and heteroplasmic substitutions in human mitogenomes. Material and methods: Mitogenomes of five samples were amplified with Long PCR Enzyme Mix from Fermentas or TaKaRa LA Taq DNA Polymerase from TaKaRa. Then, NexteraTM XT DNA libraries were sequenced on MiSeq FGx platform (Illumina). mtDNA substitutions were called for alternative variants above the 1% level. Results: All homoplasmic substitutions detected in amplicons generated with polymerases studied here and sequenced on MiSeq FGx system were consistently identified as homoplasmies with alternative sequencing methods. TaKaRa LA Taq DNA Polymerase was found to be less accurate in low-level heteroplasmy detection than Long PCR Enzyme Mix enzyme as more false negative and false positive results were observed for minority variants called above the 1% level. Nevertheless, both PCR systems studied can be successfully used to detect authentic mtDNA substitutions, for which minority variants exceed the 3.61% level assuming at least 10,000x coverage and sequencing Nextera XT DNA libraries on MiSeq FGx machine. Conclusions: The accuracy and sensitivity of point heteroplasmy detection with the MiSeq FGx instrument varies on polymerase used for mtDNA amplification. Therefore, it is recommended to validate the laboratory protocols used for mtDNA substitution detection prior to their implementation for the forensic or medical genetics purposes.

A molecular dynamics investigation of Taq DNA polymerase and its complex with a DNA substrate using a solid-state nanopore biosensor.

Proteins have a small volume difference by the diversity of amino acids, which make protein detection and identification a great challenge. Solid-state nanopore as label-free biosensors has attracted attention with high sensitivity. In this work, we investigated the Taq DNA polymerase before and after combining it with a DNA substrate on a solid-state nanopore through molecular dynamics. In simulation, we analyzed the contribution source of nanopore current blockage. In addition to considering the traditional physical exclusion volume model, the non-covalent interaction between the protein molecules and the pore wall also showed to affect the current blockage in the nanopore. When choosing pores of comparable size to protein molecules, the two states of Taq DNA polymerase produce differentiated non-covalent interactions with the pore wall, which enhanced the amplitude difference in current blockage. As a result, the two DNA polymerases can be distinguished through the distinct current blockage. However, when applying additional pulling force or increasing the pore size of the nanopore, the differences between the current blockages are not significant enough to distinguish. The introduction of the non-covalent interaction makes it clear to understand the current blockage differences, which guide the mechanism between molecules with similar structures or volumes.

Replicative bypass studies of l-deoxyribonucleosides in Vitro and in E. coli cell.

L-nucleosides were the most important antiviral lead compounds because they can inhibit viral DNA polymerase and DNA synthesis of many viruses, whereas they may lead to mutations in DNA replication and cause genomic instability. In this study, we reported the replicative bypass of L-deoxynucleosides in recombinant DNA by restriction enzyme-mediated assays to examine their impact on DNA replication in vitro and in E. coli cells. The results showed that a template L-dC inhibited Taq DNA polymerase reaction, whereas it can be bypassed by Vent (exo-) DNA polymerase as well as in cell replication, inserting correct nucleotides opposite L-dC. L-dG can be bypassed by Taq DNA polymerase and in E. coli cells, maintaining insertion of correct incoming nucleotides, and L-dG induced mutagenic replication by Vent (exo-) DNA polymerase. In contrast, L-dA can induced mutagenic replication in vitro and in E. coli cells. MD simulations were performed to investigate how DNA polymerase affected replicative bypass and mutations when D-nucleosides replaced with L-nucleosides. This study will provide a basis for the ability to assess the cytotoxic and mutagenic properties of the L-nucleoside drugs.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Reverse transcriptase-free detection of viral RNA using Hemo Klentaq DNA polymerase.

COVID-19 pandemic highlighted the demand for the fast and reliable detection of viral RNA. Although various methods for RNA amplification and detection have been proposed, some limitations, including those caused by reverse transcription (RT), need to be overcome. Here, we report on the direct detection of specific RNA by conventional polymerase chain reaction (PCR) requiring no prior RT step. It was found that Hemo KlenTaq (HKTaq), which is posed as DNA-dependent DNA polymerase, possesses reverse transcriptase activity and provides reproducible amplification of RNA targets with an efficiency comparable to common RT-PCR. Using nasopharyngeal swab extracts from COVID-19-positive patients, the high reliability of SARS-CoV-2 detection based on HKTaq was demonstrated. The most accurate detection of specific targets are provided by nearby primers, which allow to determine RNA in solutions affected to multiple freeze-thaw cycles. HKTaq can be used for elaboration of simplified amplification techniques intended for the analysis of any specific RNA and requiring only one DNA polymerase.