Large Fragment Of DNA Polymerase Research Articles

Double domain fusion improves the reverse transcriptase activity and inhibitor tolerance of Bst DNA polymerase

To enhance the DNA/RNA amplification efficiency and inhibitor tolerance of Bst DNA polymerase, four chimeric Bst DNA polymerase by fusing with a DNA-binding protein Sto7d and/or a highly hydrophobic protein Hp47 to Bst DNA polymerase large fragment. One of chimeric protein HpStBL exhibited highest inhibitor tolerance, which retained high active under 0.1 U/μL sodium heparin, 0.8 ng/μL humic acid, 2.5× SYBR Green I, 8 % (v/v) whole blood, 20 % (v/v) tissue, and 2.5 % (v/v) stool. Meanwhile, HpStBL showed highest sensitivity (93.75 %) to crude whole blood infected with the African swine fever virus. Moreover, HpStBL showed excellent reverse transcriptase activity in reverse transcription loop-mediated isothermal amplification, which could successfully detect 0.5 pg/μL severe acute respiratory syndrome coronavirus 2 RNA in the presence of 1 % (v/v) stools. The fusion of two domains with different functions to Bst DNA polymerase would be an effective strategy to improve Bst DNA polymerase performance in direct loop-mediated isothermal amplification and reverse transcription loop-mediated isothermal amplification detection, and HpStBL would be a promising DNA polymerase for direct African swine fever virus/severe acute respiratory syndrome coronavirus 2 detection due to simultaneously increased inhibitor tolerance and reverse transcriptase activity.

Production of Bst polymerase for diagnosis of different infections using loop-mediated isothermal amplification

Introduction. The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) possesses 5'-3' DNA polymerase activity, 5'-3' displacement activity and high processivity. These properties make it possible to use Bst DNA polymerase in loop-mediated isothermal amplification (LAMP), which provides highly specific amplification of the target sequence and is used for rapid detection of agents causing human infectious diseases.
 The purpose of the study was to produce a recombinant Bst polymerase enzyme in the bacterial expression system and to assess its properties for LAMP-based diagnostics of infectious diseases.
 Materials and methods. Expression constructs carrying the Bst polymerase gene were obtained using genetic engineering techniques. Different Escherichia coli strains were used for protein expression. Metal-chelate and gel filtration chromatography techniques were used for protein purification. Catalytic characteristics of the enzyme were assessed in loop-mediated isothermal amplification reactions using AmpliSens SARS-CoV-2-IT, AmpliSens IAV-IT and AmpliSens IBV-IT diagnostic systems designed for high-quality detection of SARS-CoV-2, influenza A virus (IAV) and influenza B virus (IBV) RNA, respectively.
 Results. The offered protocol for production, extraction and purification of recombinant Bst polymerase makes it possible to produce the enzyme in the bacterial expression system using E. coli cells in a soluble form and reaching the yield up to 20% of the total cell mass. In LAMP reactions, the obtained enzyme demonstrates activity comparable with that of the commercial enzyme Bst 2.0 (NEB).
 Conclusion. Considering the fast purification and production of the enzyme, the obtained recombinant Bst polymerase can be used in LAMP-based diagnostic kits.

Cloning and purification of large fragment of DNA polymerase I from Geobacillus stearothermophilus and it’s application in isothermal DNA amplification

Cloning and purification of large fragment of DNA polymerase I from Geobacillus stearothermophilus and it’s application in isothermal DNA amplification

Bst DNA Polymerase Large Fragment for Whole Genome Amplification

Bst DNA Polymerase Large Fragment for Whole Genome Amplification

Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site.

The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas.

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA

AbstractIncorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer extension reactions are commonly used for the assay of such reaction events. However, current assay protocols generally rely on radiolabeling, fluorescence reporter labeling, or removal of specific deoxynucleotide triphosphate in the reaction mixture. Herein we report on the design of two novel assay protocols that utilize a dideoxynucleotide‐terminated template strand and a phosphorothiolate‐modified deoxynucleotide‐terminated template strand. We designed and synthesized a deoxyuridine triphosphate analogue (dU*TP) containing 2‐bromoisobutyryl group and demonstrated that it could be well recognized by ϕ29DNA polymerase, E. coli DNA polymerase I Klenow Fragment, Bst DNA polymerase Large Fragment, and E. coli DNA polymerase I Klenow Fragment (exo(), which translated to effective incorporation of dU*TP into DNA. dU*TP was also successfully incorporated into extremely long single‐stranded DNA at high‐density using ϕ29 DNA polymerase by rolling circle amplification.

Establishment of a sensitive and specific hyper-branched rolling circle amplification assay and test strip for TSV

A specific padlock probe (PLP) and detection probe were designed to target the capsid protein gene of Taura syndrome virus (TSV), and a hyper-branched rolling circle amplification (HRCA) assay and a corresponding strip-based test were produced. The reaction time and temperature were optimized for both. The PLPs were linked with the target sequence via T4 DNA ligase under conditions of 37°C for 30min, followed by reaction with Bst DNA polymerase Large Fragment at 61°C for 30min, and then the test strip was made using the detection probe. Both the assay and the strip had similarly high accuracy and specificity, and their sensitivity was close to 10 copies, which is 100 times higher than that of conventional RT-PCR. We tested 89 batches of shrimps for TSV to assess the HRCA assay and test strip; the results indicated that the TSV HRCA assay and the test strip are rapid diagnostic tools for detection in the field, and have potential for early diagnosis of TSV.

Time Dependence of Partitoning between Polymerization and Editing Sites by Klenow Polymerase

Klenow polymerase is the large fragment of DNA polymerase I from E. coli. Klenow possesses 5'-3' polymerase and intrinsic 3'-5' exonuclease activities with two distinct active sites that are located ∼30 Ǻ apart. During DNA replication, proofreading activity enhances replication fidelity by excising misincorporated nucleotides from the 3' end of primer strand. The first step of the proofreading process is sending the 3'-primer terminus to the proofreading site, which requires separation of the primer terminus from the template strand. Here we examined the binding of Klenow to matched and mismatched primed-template DNA (pt-DNA) by monitoring the steady state fluorescence intensity change of a single 2-aminopurine base site-specifically substituted in the template strand within the duplex part of pt-DNA. The changes in fluorescence intensity allow us to follow shuttling of the primer terminus between the polymerization and proofreading sites.We have found that the rate of partitioning of the primer between the two active sites depends on: 1) the number of mismatched bases at the primer-template junction, 2) the presence or absence of divalent ions, and 3) the type of divalent ions. Magnesium and calcium ions have opposite effects on the direction of the shift between the pol and exo sites. Substitution of the normal phosphodiester linkage between the last two bases of the primer strand with a non-hydrolysable phosphorothioate linkage also has significant effects on the partitioning between sites.

Reverse‐Transcriptase Loop‐Mediated Isothermal Amplification as a Rapid Screening/Monitoring Tool for Salmonella Enterica Detection in Liquid Whole Eggs

Reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is a novel molecular detection method that is specific, fast, and simple. It is based on reverse transcription followed by DNA amplification using the Bst DNA polymerase large fragment requiring one temperature and a simple waterbath, without the need for any expensive equipment. Detection is by turbidity or agarose gel electrophoresis. Our objective was to apply this LAMP-based technology to rapidly and sensitively detect Salmonella enterica serovar Enteritidis in liquid whole eggs (LWEs) within 1 d. LWE were inoculated with S. Enteritidis and stomached in tetrathionate broth (TTB), and spread-plated on Xylose lysine tergitol 4 agar either immediately or after 6, 12, or 16-h enrichment. RNA was extracted from 5-mL TTB and the RT-LAMP assay was carried out using invA primers. After 16 and 12-h enrichment, improved Salmonella detection up to 10⁰ to 10¹ and 10⁴ CFU/25 mL LWE, respectively was obtained. Without enrichment, Salmonella could be detected at 10⁷ CFU/25 mL; however, after 6-h enrichment a 1-log improvement to 10⁶ CFU/25 mL was obtained. This RT-LAMP assay appears to be suitable as a potential screening/monitoring tool for Salmonella enterica from LWE products in routine settings with results obtainable within 24-h, which is significantly faster than traditional cultural assays.

Time Dependence of Switching Between Polymerization and Editing Mode DNA Binding by Klenow Polymerase

Klenow polymerase is the large fragment of DNA polymerase I from E. coli. Klenow possesses 5’-3’ polymerase and intrinsic 3’-5’ exonuclease activities with two distinct active sites that are located ∼30 angstroms apart. During DNA replication, proofreading activity enhances replication fidelity by excising misincorporated nucleotides from the 3’ end of primer strand. The first step of the proofreading process is sending the 3’-primer terminus to the proofreading site, which requires separation of the primer terminus from the template strand. Here we examined the binding of Klenow to matched and mismatched primed-template DNA (pt-DNA) by monitoring the steady state fluorescence intensity change of a single 2-aminopurine base site-specifically substituted in the template strand within the duplex part of pt-DNA. The changes in fluorescence intensity allow us to follow shuttling of the primer terminus between the polymerization and proofreading sites. We have found that partitioning of the primer between the two active sites depends on: 1) the number of mismatched bases at the primer-template junction, 2) the presence or absence of divalent ions, 3) the type of divalent ions, and 4) the time in the presence of the divalent ions. A significant time dependence of the partitioning between the two sites was observed with the matched and single mismatched pt-DNA in the presence of Mg2+. The slow kinetic dependence of the primer partitioning between the polymerization and proofreading sites in Klenow polymerase helps explain previous conflicting reports of the equilibrium partitioning between sites.

Rapid and simple detection of Hepatitis C virus by reverse transcriptase -loop- mediated isothermal amplification method

Hepatitis C virus (HCV) infection frequently causes chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. HCV infection has now become a serious health problem because at least 170 million people worldwide are currently infected with HCV. Molecular diagnostics are revolutionizing the clinical practice of infectious disease. Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification. This simple and rapid technique relies on strand-displacing DNA synthesis performed using the Bst DNA polymerase large fragment under isothermal conditions in the temperature range of 60–65°C, thereby obviating the need for a thermal cycler. The method has also been rendered applicable to RNA genomes by combining it with reverse transcription reactions (RT-LAMP). RT-LAMP is characterized by the use of 6 different primers. For 110 quantities sera, Nested RT-PCR and RT-LAMP were done. At the end of theLAMP reaction, SYBR Green was used for identifying negative and positive results. The PCR sensitivity up to 80 particles was observed and the LAMP sensitivity test was verified up to 8 particles. The data presented in this study suggested that the RT-LAMP assay is more sensitive than RT-PCR by picking up 5 additional cases that were negative by RT-PCR. These findings demonstrate that detection of HCV using RT-LAMP was more sensitive than the Nested RT-PCR. The RT-LAMP assay developed in this study is simple, rapid, and cost effective as well as highly sensitive and specific. Key words: Hepatitis C virus, RT-LAMP, nested RT-PCR.

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop-mediated isothermal amplification

The objective of this study is to develop a serovar-specific loop-mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real-time polymerase chain reaction (FQ-PCR). The results were as follows. (1) Serovar-specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl(-1) ; thus, the sensitivity and specificity of this assay is similar to those of the FQ-PCR. LAMP is a high-throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. This is the first study involving the use of LAMP to detect Salmonella serovar-specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.

A simple and rapid method for detection of Goose Parvovirus in the field by loop-mediated isothermal amplification

BackgroundGoose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, in a worldwide scale, GPV severely affects geese production. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the sensitive, rapid, and inexpensive detection of GPV in the field.ResultsA set of six specific primers was designed by targeting the GPV VP3 DNA. With Bst DNA polymerase large fragment, the target DNA could be amplified at 65°C as early as 20 min of incubation in a simple water bath. A positive reaction was identified through the detection of the LAMP product by color change visible to the naked eye. The detection limit of the assay was 28 copies/μl of plasmid pVP3, and with equal sensitivity and specificity to fluorescent quantitative real-time PCR (FQ-PCR).ConclusionsThe high sensitivity, specificity, and simplicity, as well as the high throughput, make this method suitable for specific detection of GPV infection in both field conditions and laboratory settings. The utilization of complicated equipment and conduct of technical training on the GPV LAMP were not necessary.

Efficiency and specificity of microRNA-primed nucleotide analog incorporation by various DNA polymerases

MicroRNAs (miRNAs) as endogenous regulators of gene expression have spurred a surge of interest for their quantification and expression analysis. High-sensitivity and high-specificity miRNA detection techniques, such as real-time polymerase chain reaction and recently introduced bioluminescent miRNA detection, require systematic study of DNA polymerases for use with miRNAs. In this study, a variety of DNA polymerases were studied to assess their capabilities of using miRNA as a primer and incorporating 2′-deoxyadenosine-5′- O-(1-thiotriphosphate) as a dATP alternative during DNA strand extension. Five DNA polymerases were investigated: mesophilic DNA polymerase I large (Klenow) fragment, 3′ → 5′ exo − Klenow DNA polymerase, thermophilic Bst DNA polymerase large fragment, Therminator DNA polymerase, and Taq DNA polymerase. The experimental results show that, except for Taq DNA polymerase, the polymerases can use miRNA as a primer and have both common and divergent properties of the nucleotide analog incorporation and miRNA discrimination. DNA polymerase I large (Klenow) fragment showed no detectable polymerization product with the thio-modified dATP as a substrate. Thermophilic Bst DNA polymerase had the highest specificity for miRNA recognition on a DNA template. The study provides a novel method for miRNA detection without reverse transcription to complementary DNA that is faster, simpler, and less prone to biases and errors.

Estrogen Induces Estrogen-related Receptor α Gene Expression and Chromatin Structural Changes in Estrogen Receptor (ER)-positive and ER-negative Breast Cancer Cells

Estrogen-related receptor alpha (ERRalpha), a member of the nuclear receptor superfamily, is closely related to the estrogen receptors (ERalpha and ERbeta). The ERRalpha gene is estrogen-responsive in several mouse tissues and cell lines, and a multiple hormone-response element (MHRE) in the promoter is an important regulatory region for estrogen-induced ERRalpha gene expression. ERRalpha was recently shown to be a negative prognostic factor for breast cancer survival, with its expression being highest in cancer cells lacking functional ERalpha. The contribution of ERRalpha in breast cancer progression remains unknown but may have important clinical implications. In this study, we investigated ERRalpha gene expression and chromatin structural changes under the influence of 17beta-estradiol in both ER-positive MCF-7 and ER-negative SKBR3 breast cancer cells. We mapped the nucleosome positions of the ERRalpha promoter around the MHRE region and found that the MHRE resides within a single nucleosome. Local chromatin structure of the MHRE exhibited increased restriction enzyme hypersensitivity and enhanced histone H3 and H4 acetylation upon estrogen treatment. Interestingly, estrogen-induced chromatin structural changes could be repressed by estrogen antagonist ICI 182 780 in MCF-7 cells yet were enhanced in SKBR3 cells. We demonstrated, using chromatin immunoprecipitation assays, that 17beta-estradiol induces ERRalpha gene expression in MCF-7 cells through active recruitment of co-activators and release of co-repressors when ERRalpha and AP1 bind and ERalpha is tethered to the MHRE. We also found that this estrogen effect requires the MAPK signaling pathway in both cell lines.

Rapid and sensitive detection of Singapore grouper iridovirus by loop-mediated isothermal amplification

The aim of this paper was to develop a loop-mediated isothermal amplification (LAMP) method for rapid, sensitive and inexpensive detection of Singapore grouper iridovirus (SGIV) in grouper (GP), Epinephelus sp. A set of six specific primers was designed by targeting the SGIV ORF-014L. With Bst DNA polymerase large fragment, the target DNA can be amplified as early as 20 min at 65 degrees C in a simple water bath. The detection limit is about 0.02 fg (equivalent to 6.3 copies) of plasmid ORF-014L. LAMP products could be judged with three different methods. There were no cross-reactions with seven other aquatic animal viruses indicating high specificity of the LAMP. The LAMP method was applied to detect SGIV in virus-infected GP cells and GP tissues effectively. The LAMP described in this study is a cheap, sensitive, specific and rapid protocol for the detection of SGIV in cells and in GP tissues. The developed LAMP method can be simply applied both in field condition and in laboratory operation for specific detection of SGIV infection.

Dlx Homeobox Genes Promote Cortical Interneuron Migration from the Basal Forebrain by Direct Repression of the Semaphorin Receptor Neuropilin-2

Dlx homeobox genes play an important role in vertebrate forebrain development. Dlx1/Dlx2 null mice die at birth with an abnormal cortical phenotype, including impaired differentiation and migration of GABAergic interneurons to the neocortex. However, the molecular basis for these defects downstream of loss of Dlx1/Dlx2 function is unknown. Neuropilin-2 (NRP-2) is a receptor for Class III semaphorins, which inhibit neuronal migration. Herein, we show that Neuropilin-2 is a specific DLX1 and DLX2 transcriptional target by applying chromatin immunoprecipitation to embryonic forebrain tissues. Both homeobox proteins repress Nrp-2 expression in vitro, confirming the functional significance of DLX binding. Furthermore, the homeodomain of DLX1 and DLX2 is necessary for DNA binding and this binding is essential for Dlx repression of Nrp-2 expression. Of importance, there is up-regulated and aberrant expression of NRP-2 in the forebrains of Dlx1/Dlx2 null mice. This is the first report that DLX1 or DLX2 can function as transcriptional repressors. Our data show that DLX proteins specifically mediate the repression of Neuropilin-2 in the developing forebrain. As well, our results support the hypothesis that down-regulation of Neuropilin-2 expression may facilitate tangential interneuron migration from the basal forebrain.

A novel one cycle allele specific primer extension—Molecular beacon displacement method for DNA point mutation detection with improved specificity

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3′ (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the “wrong” genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3′ exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3′-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3′-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.

A Chromatin-associated and Transcriptionally Inactive p53-Mdm2 Complex Occurs in mdm2 SNP309 Homozygous Cells

In cancer cells, the function of the tumor suppressor protein p53 is usually blocked. Impairment of the p53 pathway results in tumor cells with endogenous overexpression of Mdm2 via a naturally occurring single nucleotide polymorphism (SNP) in the mdm2 gene at position 309. Here we report that in mdm2 SNP309 cells, inactivation of p53 results in a chromatin-associated Mdm2-p53 complex without clearance of p53 by protein degradation. Nuclear accumulation of p53 protein in mdm2 SNP309 cells results after 6 h of camptothecin, etoposide, or mitomycin C treatment, with the p53 protein phosphorylated at Ser15. Chromatin immunoprecipitation demonstrated p53 and Mdm2 bound to p53 responsive elements. Interestingly, although the p53 protein was able to bind to DNA, quantitative PCR showed compromised transcription of endogenous target genes. Additionally, exogenously introduced p53 was incapable of activating transcription from p53 responsive elements in SNP309 cells, confirming the trans-acting nature of the inhibitor. Inhibition of Mdm2 by siRNA resulted in transcriptional activation of these p53 targets. Our data suggest that overproduction of Mdm2, resulting from a naturally occurring SNP, inhibits chromatin-bound p53 from activating the transcription of its target genes.

Crystal structures of a ddATP-, ddTTP-, ddCTP, and ddGTP- trapped ternary complex of Klentaq1: insights into nucleotide incorporation and selectivity.

The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP). In this report, we present the details of the 2.3-A resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP), or a dideoxyadenosine 5'-triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson-Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen-bonding interactions may provide a means of detecting misincorporation at this position.