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At least 19 recordsLinked to original sources
PubMed1997

Primer/template-independent synthesis of poly d(A-T) by Taq polymerase.

Taq DNA polymerase polymerized dATP and dTTP to poly d(A-T) without requiring added primer/template in the temperature range of 60-70 degrees C. Tth DNA polymerase also catalyzed the reaction, while delta Tth, Vent, Vent(exo-), Pfu, Ultma, BcaBEST, and KOD DNA polymerases did not. The reaction was distinct from the template-nonrequiring terminal deoxynucleotidyl transferase reaction which absolutely required primers.

DNA Nucleotidylexotransferase↗
PubMed1992

Amplimers with 3'-terminal phosphorothioate linkages resist degradation by vent polymerase and reduce Taq polymerase mispriming.

The 3'-->5' exonuclease activity of Vent, a thermostable polymerase from Thermococcus litoralis, enhances DNA replication fidelity but also diverts PCR primers (amplimers) from targeted amplification by degrading their 3' termini. We demonstrate that amplimers with a 3-base 3'-terminal mismatch can be efficiently truncated by Vent to prime DNA polymerizations that compete with the specific amplification reaction. However, amplimers with phosphorothioate bonds joining their 3'-terminal residues are resistant to degradation and demonstrate greatly enhanced priming specificity. Slight destabilization of base-pairing by phosphorothioate bond-linked residues also diminishes extension of mispaired 3' amplimer termini in Taq polymerase-mediated amplifications.

Bacterial Proteins↗
PubMed1996

Structure of Taq polymerase with DNA at the polymerase active site.

The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologous to Escherichia coli DNA polymerase I (Pol I) and likewise has domains responsible for DNA polymerase and 5' nuclease activities. The structures to the polymerase domains of Taq polymerase and of the Klenow fragment (KF) of Pol I are almost identical, whereas the structure of a vestigial editing 3'-5' exonuclease domain of Taq polymerase that lies between the other two domains is dramatically altered, resulting in the absence of this activity in the thermostable enzyme. The structures have been solved for editing complexes between KF and single-stranded DNA and for duplex DNA with a 3' overhanging single strand, but not for a complex containing duplex DNA at the polymerase active-site. Here we present the co-crystal structure of Taq polymerase with a blunt-ended duplex DNA bound to the polymerase active-site cleft; the DNA neither bends nor goes through the large polymerase cleft, and the structural form of the bound DNA is between the B and A forms. A wide minor groove allows access to protein side chains that hydrogen-bond to the N3 of purines and the O2 of pyrimidines at the blunt-end terminus. Part of the DNA bound to the polymerase site shares a common binding site with DNA bound to the exonuclease site, but they are translated relative to each other by several angstroms along their helix axes.

Binding Sites↗
PubMed2004

Structure-specific DNA-induced conformational changes in Taq polymerase revealed by small angle neutron scattering.

The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5'-nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNA-bound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.

Base Sequence↗
PubMed1992

Reverse transcriptase inhibits Taq polymerase activity.

Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.

Avian Myeloblastosis Virus↗
PubMed1989

Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing.

Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.

Base Sequence↗
PubMed2004

The implications of using mutagenic primers in combination with Taq polymerase having proofreading activity.

Polymerases with proofreading activity provide high fidelity PCR amplifications. In this study we examined the consequences of using a Taq polymerase with proofreading activity, such as Optimase Taq polymerase, in combination with 4 different mutagenic reverse primers for the amplification of a 345-bp FII PCR product. The amplifications were performed with Optimase Taq polymerase (Transgenomic), and Taq DNA polymerase-recombinant (Invitrogen), without proofreading activity. Mutation screening was carried out by DHPLC and restriction fragment analysis. The usage of Optimase Taq polymerase results in complete reversion of the first and second mutated nucleotide introduced at the 3' end of the mutagenic reverse primer. It also partially reverses the missense nucleotide introduced in the third position of the mutagenic primer and leads to misleading DHPLC and restriction fragment analysis patterns. Nevertheless it cannot perform such an activity when an abnormal nucleotide is introduced in the fourth position.

Base Sequence↗
PubMed2005

Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid.

AIM: To investigate the dose-response effect of humic acid (HA) on the quantitative real time polymerase chain reaction (QRT-PCR) inhibition and the efficiency of Taq polymerase increment in preventing inhibition by HA in DNA extracted from ancient bones. METHODS: DNA was isolated from bone samples and DNA quantification was conducted with the real-time 5' exonuclease detection assay (TaqMan), using the ABI PRISM 7000 instrument. RESULTS: The addition 10-75 ng of synthetic HA inhibited QRT-PCR, whereas the addition of 100 ng of synthetic HA completely inhibits QRT-PCR. The addition of 1.25 Unit (U) of Taq polymerase per assay appeared to be the optimum amount in overcoming the HA inhibition. The best results were obtained when crude DNA extracts containing humic substances were quantified by QRT-PCR with the addition of 1.25 Unit (U) of extra Taq polymerase per assay. CONCLUSION: The modified procedure with increased Taq polymerase concentration should allow more effective QRT-PCR analysis in samples containing HA.

Bone and Bones↗
PubMedDate not supplied

Osmoregulated TAQ polymerase gene expression in Escherichia coli.

The Thermus aquaticus DNA Polymerase I (Taq Pol I) gene was cloned into the pOSEX4 plasmid under the osmo-inducible promoter proU and subsequently expressed into the Escherichia coli MKH13 strain. The suitability of the enzyme in polymerase assays was determined in standard 35S dATP incorporation tests and by PCR. The Taq Pol I expression in this system, which is under the control of the osmotic pressure in the growth medium, was analyzed in different media and in different sodium chloride concentrations. A study of the osmolarity effects in the growth of the strain and in Taq Pol I expression shows that an increase in sodium chloride concentration limits the growth. At 0.25 M of NaCl maximum activity was observed; at higher values of osmolarity, we found an unexpected decline of activity. This is the first report of using the pOSEX vector for the expression of an heterologous protein and it is very advantageous to make a regulated, non toxic, simple and cost-effective manner of induction in a biotechnology process using just NaCl or other non-permeable osmolyte.

Biotechnology↗
PubMed1989

An improved method for photofootprinting yeast genes in vivo using Taq polymerase.

We have developed an improved method for photofootprinting in vivo which utilizes the thermostable DNA polymerase from T. aquaticus (Taq) in a primer extension assay. UV light is used to introduce photoproducts into the genomic DNA of intact yeast cells. The photoproducts are then detected and mapped at the nucleotide level by multiple rounds of annealing and extension using Taq polymerase, which is blocked by photoproducts in the template DNA. The method is more rapid, sensitive, and reproducible than the previously described chemical photofootprinting procedure developed in this laboratory (Nature 325. 173-177), and detects photoproducts with a specificity which is similar, but not identical to that of the previously described procedure. Binding of GAL4 protein to its binding sites within the GAL1-10 upstream activating sequence is demonstrated using the primer extension photofootprinting method. The primer extension assay can also be used to map DNA strand breakage generated by other footprinting methods, and to determine DNA sequence directly from the yeast genome.

Base Sequence↗
PubMed1990

Reverse transcription and direct amplification of cellular RNA transcripts by Taq polymerase.

We report the ability of Taq polymerase to directly transcribe RNA templates in vitro. We have made use of this finding to develop a single-step protocol for amplification of RNA transcripts. The method was shown to require only subnanogram amounts of total cellular RNA as starting material. A microassay was developed in which RNA can be extracted from one drop of blood or 1000 cultured cells, and analyzed for the expression of a specific gene.

Base Sequence↗
PubMed1997

Mutagenesis of the positively charged conserved residues in the 5' exonuclease domain of Taq DNA polymerase.

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method. Taq DNA polymerase has a domain at the amino terminus (residue 1 to 290) that has a 5' exonuclease activity and a domain at the C-terminus that catalyzes polymerase reaction. Taq DNA polymerase is classified into the pol I family which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5' exonuclease domains of the pol I family DNA polymerases shows six highly conserved sequences called motifs A to F. Motif C contains three positively charged residues such as 74Arg, 82Lys and 85Arg which might be involved in catalysis. In order to understand the function of those residues, they are mutagenized to alanine. The 5' exonucleolytic activities of those mutated 5' exonucleases decreased by 80 to 90%, thereby implying that three positively charged residues play certain roles in the 5' exonuclease catalysis.

Amino Acid Sequence↗
PubMed2006

The occurrence of antibiotic resistance genes in Taq polymerases and a decontamination method applied to the detection of genetically modified crops.

Different antibiotic resistance (AR) genes, such as Bla, Tet and NPTII, contaminate commercially available Taq polymerases. The specificity of the AR gene PCR can be increased when using a restriction enzyme-based decontamination of polymerase. The elimination of Taq polymerase contamination allows the use of PCR tests to screen seeds (corn) and processed food for the presence of genetically modified organisms (GMO) based on the detection of AR genes. Without a decontamination procedure for AR genes, PCR screening tests should be interpreted with caution.

Crops, Agricultural↗
PubMed2004

Experimental procedures comparing the activity of different Taq polymerases.

Forensic investigations involve several scientific branches among which biological analyses are much more frequently requested as a consequence of their importance and great versatility towards most of the traces found on the crime scene. Biological analyses are lead in subsequent steps: extraction, amplification and STR typing of the specimens collected on the crime scene. All of these techniques have been modified from original protocols according to the kind of sample to process. A critical point in our analysis is trying to amplify small amounts of DNA extracted from decomposed tissues or objects, small biological traces have been left on, with high fidelity and account. That's why we have decided to settle on an experimental procedure aimed to find the best DNA polymerase according to our purposes. We have tested different Taq polymerases on the same known DNA sample at several dilutions and have compared quality and amount of amplified DNA in order to appreciate the amplifying capability of each enzyme. These data have been analyzed as a function of the technical properties of each engineered Taq polymerase and results are shown in details.

DNA↗
PubMed1999

Enrichment polymerase chain reaction for the detection of Ki-ras mutations: relevance of Taq polymerase error rate, initial DNA copy number, and reaction conditions on the emergence of false-positive mutant bands.

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

False Positive Reactions↗
PubMed2005

Coupled protein domain motion in Taq polymerase revealed by neutron spin-echo spectroscopy.

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

Models, Molecular↗
PubMed1997

Production and evaluation of Taq DNA polymerase.

Taq DNA polymerase is an enzyme essential in performing Polymerase Chain Reaction (PCR) which has recently become a basic technology in research and diagnostic laboratories. In order to reduce the cost of research work in Thailand, recombinant Taq DNA polymerase was locally produced from pTaq cloned in E. coli. The enzyme was characterized and evaluated in comparison with the commercial Taq DNA polymerase produced by Perkin Elmer Cetus, U.S.A. The yield of enzyme was 6.72 mg/ml and the activity of 9,524 units/mg protein with the total of 448,000 units/litre of the bacterial culture. The preparation was free of DNase based upon its ability to degrade Lambda DNA evaluated by gel electrophoresis. Although the enzyme produced gave a high DNA polymerase activity, the preparation was not as pure as the enzyme produced by Perkin Elmer Cetus. Immunoblot analysis indicated that the enzyme preparation contained the products of enzyme degradation obtained during preparation and bacterial protein contaminations. In spite of the existence of bacterial proteins in the preparation, the Taq enzyme produced was proved to be applicable in performing PCR such as the PCR-SSP (Sequence Specific Primers) typing for HLA-DR. The cost of enzyme preparation was about 256 times less than that of the commercial enzyme. Economically, the locally produced Taq DNA polymerase can be used efficiently in the research laboratories performing PCR based typing of the HLA genes.

Analysis of Variance↗