Heat-mediated activation of affinity-immobilized Taq DNA polymerase

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.     

Photoaffinity labeling of DNA polymerase from Thermus thermophilus and DNA template by photoreactive analogs of dCTP

The aim of this study was to evaluate the influence of nicotine on the daily rhythms of heart rate, body temperature and locomotor activity in unrestrained rats by use of implanted radiotelemetry transmitters. The study was divided into three seven-day periods: a control period, a treatment period and a recovery period. The control period was used for baseline measurement of heart rate, body temperature and locomotor activity. During the treatment period three rats received nicotine (1 mg kg(-1), s.c.) at 0900 h. Three rats received saline under the same experimental conditions. Heart rate, body temperature and locomotor activity were continuously monitored and plotted every 10 min. During the three periods a power spectrum analysis was used to determine the dominant period of rhythmicity. If daily rhythms of heart rate, body temperature and locomotor activity were detected, the characteristics of these rhythms, i.e. the mesors, amplitudes and acrophases, were determined by cosinor analysis, expressed as means +/- s.e.m. and compared by analysis of variance. Nicotine did not suppress daily rhythmicity but induced decreases of amplitudes and phase-advances of acrophases for heart rate, body temperature and locomotor activity. These perturbations might result from the effects of nicotine on the suprachiasmatic nucleus, the hypothalamic clock that co-ordinates biological rhythms.     

Two different reactions involved in the primer/template-independent polymerization of dATP and dTTP by Taq DNA polymerase

We previously isolated a 41-kDa early antigen of human herpesvirus 6 (HHV-6), which exhibited nuclear localization and DNA-binding activity (Agulnick et al., 1993). In this study, we observed that a 110-kDa protein was coimmunoprecipitated with p41 from HHV-6-infected cells by an anti-p41 antibody. This 110-kDa protein was identified as the HHV-6 DNA polymerase (Pol-6) by an antibody raised against the N terminus of Pol-6. Reciprocal immunoprecipitation and Western blot analyses confirmed that p41 complexes with Pol-6 in HHV-6-infected cells. In addition, both p41 and Pol-6 were expressed in vitro and shown to form a specific complex. An in vitro DNA synthesis assay using primed M13 single-stranded DNA template demonstrated that p41 not only increased the DNA synthesis activity of Pol-6 but also allowed Pol-6 to synthesize DNA products corresponding to full-length M13 template (7249 nucleotides). By contrast, Pol-6 alone could only synthesize DNA of <100 nucleotides. The functional interaction between Pol-6 and p41 appears to be specific because they could not be physically or functionally substituted in vitro by their herpes simplex virus 1 homologues. Moreover, as revealed by mutational analysis, both the N and C termini of Pol-6 contribute to its binding to p41. In the case of p41, the N terminus is required for increasing DNA synthesis but not binding to Pol-6, whereas the C terminus is totally dispensable.     

Human DNA polymerase beta recognizes single-stranded DNA using two different binding modes

Interactions between the human DNA polymerase beta (pol beta) and a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence increase of the poly(dA) etheno-derivative (poly(depsilonA)) as a function of the binding density of pol beta-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol beta forms a complex with a ssDNA in which 16 nucleotides are occluded by the enzyme. In the second phase, transition to a complex where the polymerase occludes only 5 nucleotides occurs. Thus, human pol beta binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol beta)16 and the second as (pol beta)5 binding modes. The analyses of the enzyme binding to ssDNA have been performed using statistical thermodynamic models, which account for the existence of the two binding modes of the enzyme, cooperative interactions, and the overlap of potential binding sites. The importance of the discovery that human pol beta binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol beta, is discussed.     

DNA labeling reagent containing carbodiimide

We have synthesized a number of fluorescent compounds having a carbodiimide function and tested if it can be used to label DNA. They all reacted well with DNA in a very short time. Some of them showed different fluorescent characteristics when they bound to DNA such as band shift or change in fluorescent intensity.     

Transcriptional sequencing: A method for DNA sequencing using RNA polymerase

We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3'-deoxynucleoside triphosphate (3'-dNTP). This method enables us to conduct a rapid isothermal sequencing reaction in <30 min, to reduce the amount of template required, and to do PCR direct sequencing without the elimination of primers and 2'-dNTP, which disturbs the Sanger sequencing reaction. An accurate and longer read length was made possible by newly designed four-color dye-3'-dNTPs and mutated RNA polymerase with an improved incorporation rate of 3'-dNTP. This method should be useful for large-scale sequencing in genome projects and clinical diagnosis.     

Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase

Inadequate yields of PCR product and the generation of nonspecific PCR products can complicate genotyping studies, particularly when the DNA template is of inferior quality and/or has a low-copy number. In this study, the ability of AmpliTaq Gold DNA Polymerase to enhance the specificity and yield of amplification was evaluated in a quadruplex short tandem repeat (STR) system in which a nonspecific PCR product and poor yield had been previously observed with AmpliTaq DNA Polymerase usage. Because AmpliTaq Gold is inactive until heated during the PCR before thermal cycling, effects similar to those achieved with "hot-start" PCR were attained in a fast, simple and practical fashion. A significant enhancement in yield at the four STR loci and improved balance of alleles resulted with the use of AmpliTaq Gold. Furthermore, a non-specific PCR product, the result of mispriming, was effectively eliminated. The consistency of quality results was improved, thereby promoting successful typing of suboptimal DNA samples and enhancing the accuracy of genotyping. Since PCR product yield is elevated with AmpliTaq Gold usage, and consistent performance and low background are achieved with higher amounts of AmpliTaq Gold compared with AmpliTaq, AmpliTaq Gold can be used to augment measures taken to counteract the effects of some PCR/Taq DNA polymerase inhibitors, such as those found in blood and some forensic specimens. Studies showed that pH affects either the activity or the activation of the polymerase. AmpliTaq Gold was found to be compatible with pH 8.3 buffers, such as GeneAmp PCR Buffer and AmpFlSTR PCR Reaction Mix but not compatible with pH 9.0 buffers, such as GenePrint STR 10 x Buffer (however, conditions for the usage of AmpliTaq Gold with the GenePrint CTTv system are provided). AmpliTaq Gold is useful for the development and optimization of multiplex amplification systems, particularly those in which the primers are not well designed and/or the reaction conditions are not optimal. Finally, because AmpliTaq Gold is initially inactive, preparation of reactions at ambient temperature and automation of the PCR are facilitated. Therefore throughput can be expanded significantly with the use of AmpliTaq Gold DNA Polymerase.     

DNA polymerase of Dictyostelium discoideum

Only one molecular weight species of DNA polymerase was found in different developmental stages of the eukaryotic microorganism Dictyostelium discoideum. The molecular weight of this DNA polymerase is estimated to be about 127 000 by sucrose gradient centrifugation. The enzyme is present in all stages of growth and development, including dormant spores. All DNA polymerase activity is lost upon incubation of the crude extract with N-ethylmaleimide. The reaction properties, molecular weight and N-ethylmaleimide sensitivity of the D. discoideum DNA polymerase are similar to those of the DNA polymerase-alpha from mammalian sources.     

Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

A lung suppuration may result in a lung bulla with its own course. We report such a case following a Pseudomonas aeruginosa pneumonia of the upper right lobe, after aspiration of gastric contents, in a 21-year-old tracheotomized patient in chronic post-traumatic coma. Mechanical ventilation (IPPV) was indicated because of respiratory insufficiency. The pneumonia was followed by an abscess and later a lung bulla, increasing in size under the effect of mechanical ventilation with progressive mediastinal compression. Surgery was contraindicated because of poor physical status. An acute episode of cardiac tamponade was controlled with an emergency transthoracic drain insertion into the bulla. The course was favourable after a drainage for 23 days and a persisting small cavity in the lung apex. All weaning attempts being unsuccessful, the patient was discharged under home mechanical ventilation. A CT-scan control 6 months later showed a normal lung parenchyma. The various alternative techniques to surgery for treatment of a lung bulla are discussed.     

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a gamma turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.     

Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive polymerase chain reaction and enzyme-linked immunosorbent assay

The rising interest in gene therapy for the treatment of numerous disorders necessitates the need for the large-scale production of therapeutic biopharmaceuticals that meet stringent purity standards. Residual host cell DNA in recombinant pharmaceuticals has been identified as a potential risk factor that must be quantitated carefully both during the manufacturing process and in the final product. We describe a PCR method to quantitate contaminating levels of host cell DNA in clinical plasmid DNA preparations intended for human gene therapy. The quantitation is based on the coamplification of two similar templates, the target DNA and a synthetic competitor, and the quantitation of the resulting PCR products. The competitor is identical to the target DNA PCR product except for a 29-bp internal replacement. As a result, the two PCR products can easily be distinguished from each other. The competitive nature of the assay allows the use of the ratio of the target DNA PCR product to the competitor DNA PCR product to determine the original amount of target DNA in a sample. The primers used in this assay anneal to a conserved region of the E. coli 23S rRNA gene. One of the primers is biotinylated, allowing the PCR products to be detected colorimetrically after their capture on microtiter plates. The capture is accomplished by differential hybridization to target and competitor-specific probes covalently attached to wells of microtiter plates. The entire assay is performed in less than 2 hr postamplification. This method represents an attractive alternative to Southern blot analysis, which is the currently established method for DNA quantitation.     

Ligation mediated fluorescent labeling of DNA sequencing primers

Automated DNA sequencing utilizing fluorescently labeled primers is a proven methodology for generating quality sequence data. However, for directed primer walking strategies this necessitates synthesis and labeling of a unique primer for each sequencing reaction. Here, we describe a rapid ligation-based method of generating labeled sequencing primers. An unlabeled 5'-phosphorylated sequencing primer is ligated to a fluorescent oligonucleotide by use of a bridge primer which is complementary to portions of the previous two oligonucleotides, thus aligning them properly for ligation. The resulting fluorescent hybrid primer can be utilized directly in cycle sequencing reactions without any prior purification.     

Mitochondrial DNA polymerase from rat liver

A DNA-dependent DNA polymerase from rat liver mitochondria was partially purified and characterized. Mitochondrial DNA polymerase has been found to be quite different from other DNA-dependent DNA polymerases alpha and beta present in the rat liver in the following points: elution patterns in a DEAE-cellulose column chromatography, sedimentation coefficients determined by the glycerol gradient centrifugation in the presence of high salt, and sensitivities to N-ethylmaleimide, ethidium bromide and KCl.     

Typing of DNA HLA-DQ alpha alleles extracted from human nail material using polymerase chain reaction

The deoxyribonucleic acid (DNA) typing of human Leukocyte Antigen (HLA) DQ alpha from human fingernails is described. HLA-DQ alpha genotypes can be accurately determined from clipped fingernails. We have typed 26 nails accurately, while one did not give any type since that one sample did not amplify due to the low quantity of DNA. The cut off limit for the digested material to be amplified is approximately 9 mgs of nail material.     

A new mammalian DNA polymerase with 3' to 5' exonuclease activity: DNA polymerase delta

A new species of DNA polymerase has been purified more than 10 000-fold from the cytoplasm of erythroid hyperplastic bone marrow. This DNA polymerase, in contrast to previously described eukaryotic DNA polymerases, is associated with a very active 3' to 5' exonuclease activity. Similar to the 3' to 5' exonuclease activity associated with prokaryotic DNA polymerases, this enzyme catalyzes the removal of 3'-terminal nucleotides from DNA, as well as a template-dependent conversion of deoxyribonucleoside triphosphates to monophosphates. The exonuclease activity is not separable from the DNA polymerase activity by chromatography on DEAE-Sephadex or hydroxylapatite, and upon sucrose density gradient centrifugation the two activities cosediment at 7 S or at 11 S depending on the ionic strength. Both exonuclease and polymerase activities have identical rates of heat inactivation and both are equally sensitive to hemin and Rifamycin AF/013, inhibitors of DNA synthesis that act by binding to DNA polymerase and causing its dissociation from its template/primer. These results are consistent with the coexistence of two enzyme activities in a single protein.     

Poly(ADP-ribose) polymerase stimulates DNA polymerase alpha by physical association

The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the Vmax of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase alpha, suggesting the existence in vivo of a complex of these two enzymes.     

The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I

Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability. An insertional mutant strain, S. yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S. yanoikuyae B1. Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent. A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques. The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S. yanoikuyae B1.     

Template specificity of rat mitochondrial DNA polymerase

Mitochondrial DNA polymerase was purified 2300-fold over isolated mitochondria from rat liver. Template-primer specificities of this enzyme were investigated. Activated DNA was satisfactorily used as an active template-primer, but both native and denatured DNAs showed a slight activity. Synthetic polynucleotide, poly(dA) - oligo(dT)10 was found to have a high efficiency under the same condition for activated DNA. When the closed-circular, nicked and gapped Co1E1 DNAs were employed as a template-primer, the enzyme could only utilize the gapped DNA, indicating that the displacement synthesis was not catalyzed by the enzyme itself. The enzyme also copied poly(A) - oligo(dT)10 in high efficiency at pH 7.5 in the presence of MnCl2. Such RNA-directed DNA polymerase activity of the enzyme was further characterized. Cofractionated endouclease activity was completely separated from the enzyme by glycerol gradient centrifugation.