Thermostable DNA polymerases can perform translesion synthesis using 8-oxoguanine and tetrahydrofuran-containing DNA templates

The translesion synthesis (TLS) capacity of the thermostable DNA polymerases Taq, Tte and Tte-seq utilizing a synthetic abasic site, tetrahydrofuran (THF), and an 8-oxoguanine-containing DNA template was investigated. Measurements with human DNA polymerase beta were used as a "positive control". Thermostable DNA polymerases were observed to perform TLS with different specificities on both substrates. With a THF-containing template, dGMP was preferentially inserted by all the DNA polymerases. In the presence of Mn(II) as a cofactor, all the polymerases incorporated dCMP opposite 8-oxoguanine whereas, in the presence of Mg(II) ions, dAMP was incorporated. It was found that none of the thermophilic DNA polymerases utilized dTTP with either an 8-oxoguanine or a THF-containing template. In all cases, DNA duplex containing THF as damage was processed to full length less effectively than DNA duplex containing 8-oxoguanine.     

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase.

We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a single round of DNA synthesis of the lac Z alpha gene by Taq polymerase responds to changes in dNTP concentration, pH, and the concentration of MgCl2 relative to the total concentration of deoxynucleotide triphosphates present in the reaction. Both base substitution and frameshift error rates of less than 1/100,000 were observed at pH 5-6 (70 degrees C) or when MgCl2 and deoxynucleotide triphosphates were present at equimolar concentrations. These high fidelity reaction conditions for DNA synthesis by the Taq polymerase may be useful for specialized uses of DNA amplified by the polymerase chain reaction.     

Novel PCR-mediated mutagenesis employing DNA containing a natural abasic site as a template and translesional Taq DNA polymerase

We describe a novel method of PCR-mediated mutagenesis employing DNA containing a natural abasic site and translesional Taq DNA polymerase. This method incorporated an adenine (80.8%) or guanine (7.7%) residue or led to a base deletion mutation (11.2%) opposite the abasic site. We conclude that the combination of DNA containing an abasic site and translesional Taq DNA polymerase is an easy and useful technique for PCR-mediated mutagenesis, having advantages different from those of conventional error-prone PCR.     

Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.

The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.     

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs'' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication.     

Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase

We have determined the fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus. Using a DNA substrate that contains a 3'-OH terminal mismatch, we demonstrate that the purified polymerase lacks detectable exonucleolytic proofreading activity. The fidelity of the Taq polymerase was measured by two assays which score errors produced during in vitro DNA synthesis of the lacZ alpha complementation gene in M13mp2 DNA. In both assays, the Taq polymerase produces single-base substitution errors at a rate of 1 for each 9000 nucleotides polymerized. Frameshift errors are also produced, at a frequency of 1/41,000. These results are discussed in relation to the effects of high temperature on fidelity and the use of the Taq DNA polymerase as a reagent for the in vitro amplification of DNA by the polymerase chain reaction.     

Sequencing PCR DNA amplified directly from a bacterial colony

We show that PCR product asymmetrically amplified directly from a bacterial colony can be sequenced to yield results as good as those obtained when purified template DNA is used for the PCR amplification step. With either template, greater than 300 nucleotides can be read from a typical sequencing reaction. Taq DNA polymerase was used for both the PCR amplification and sequencing reactions.     

Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA (RAPD) technique

Abstract: Chromosomal DNA from Bacillus subtilis , bound on the clay minerals, montmorillonite (Wyoming (W) and Apache County (Ap)) and kaolinite (K), was subjected to the random amplified polymorphic DNA (RAPD) technique. DNA bound on the clays was not amplified with 0.625, 1.875, 6.25, and 12.5 U of Taq DNA polymerase, but amplification occurred when the clay-DNA complexes were diluted 10- and 20-fold or when 21 U of Taq DNA polymerase was added. DNA desorbed from the Ap-DNA and K-DNA equilibrium complexes was amplified with 0.625 U of Taq DNA polymerase, whereas amplification of DNA desorbed from the W-DNA complex occurred only after a 10-fold dilution or when 1.875 U of Taq DNA polymerase was used. These observations indicate that clay minerals differentially affect the amplification process, probably by inhibiting the activity of Taq DNA polymerase.     

DNA recombination during PCR.

PCR co-amplification of two distinct HIV1 tat gene sequences lead to the formation of recombinant DNA molecules. The frequency of such recombinants, up to 5.4% of all amplified molecules, could be decreased 2.7 fold by a 6 fold increase in Taq DNA polymerase elongation time. Crossover sites mapped essentially to three discrete regions suggesting specific Taq DNA polymerase pause or termination sites. PCR mediated recombination may be a problem when studying heterogeneous genetic material such as RNA viruses, multigene families, or repetitive sequences. This phenomenon can be exploited to create chimeric molecules from related sequences.     

Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus

The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.     

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.     

The kinetics of fluorescent DNA labeling using PCR with different Taq polymerases depends on the chemical structures of modified nucleotides

The kinetics of DNA labeling during PCR using six fluorescent derivatives of 2′-deoxyuridine 5′-triphosphate has been studied. These compounds differ in their chemical structure, total electric charge and the length of the linker between a dye and the C5 position of a pyrimidine base. The efficiency of the incorporation of the fluorescent derivatives into a growing DNA chain by four commercially available Taq DNA polymerases with 5′→3′ exonuclease and hot start activity has been determined using real-time PCR with a TaqMan probe and the subsequent electrophoretic analysis of the reaction products. Modified deoxyuridines with a total positive or negative charge of the chromophore were practically not incorporated by Taq polymerases during PCR. The modified deoxyuridines with a neutral charge of the chromophore were effectively incorporated into DNA. The extended length of the linker between the pyrimidine base and the chromophore led to a lower PCR inhibition and a more effective inclusion of modified nucleotides in the growing DNA chain. This fact can be explained by the reduced steric effects that were caused by the dye. As a result, the most promising combinations of fluorescently labeled nucleotide and Taq polymerase have been chosen for further use in fluorescent DNA labeling.     

Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR.

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.     

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.     

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.     

大肠杆菌表达的TaqDNA聚合酶的纯化

Taq DNA聚合酶是分子生物学研究中最常用的热稳定DNA聚合酶之一,与其他热稳定DNA聚合酶具有相似的特征,其纯化策略不但有潜在的应用前景,也对同类聚合酶的分离具有指导意义。已报道的适宜大量制备Taq酶的方案所需成本较高,而文章介绍了一种利用国产阳离子交换树脂廉价制备Taq酶的方案。在本方案中,采用热变性、(NH4)SO4沉淀与724离子交换层析分离大肠杆菌表达的Taq酶,约18 g Na型树脂干粉一次可回收比活约8 131.98 U/mg、总酶活2.2×105U、近27.07 mg Taq酶。纯化的产率可达48.92%,纯化倍数约59.35。所制酶SDS-PAGE电泳只检测到94 kDa单一蛋白条带,未检测到DNA核酸酶污染,与商品酶的PCR扩增能力无区别。此纯化方法成本低,适合实验室一般性的制备和生产应用。     

Incorporation of oxidized guanine nucleoside 5'-triphosphates in DNA with DNA polymerases and preparation of single-lesion carrying DNA

We investigated the incorporation of oxidatively modified guanine residues in DNA using three DNA polymerases, Escherichia coli Kf exo+, Kf exo-, and Taq DNA polymerase. We prepared nucleoside 5'-triphosphates with modified bases (dN (ox)TP) including imidazolone associated with oxazolone (dIzTP/dZTP), dehydroguanidinohydantoin (dOGhTP), and oxaluric acid (dOxaTP). We showed that the single-nucleotide incorporation of these dN (ox)TP at the 3'-end of a primer DNA strand was possible opposite C or G for dIzTP/dZTP, opposite C for dOGhTP using the Klenow fragment, and opposite C for dOxaTP using Taq. The efficiency of these misincorporations was compared to that of the nucleoside 5'-triphosphate modified with the mutagenic guanine lesion 8-oxo-G opposite A or C as well as to that of the natural dNTPs. The reaction was found not competitive. However, the ability of Kf exo- to further copy the whole template DNA strand from the primer carrying one modified residue at the 3'-end proved to be easy and rapid. The two-step polymerization process consisting of the single-nucleotide extension followed by the full extension of a primer afforded a method for the preparation of tailored double-stranded DNA oligonucleotides carrying a single modified base at a precise site on any sequence. This very rapid method allowed the incorporation of unique residues in DNA that were not available before due to their unstable character.     

Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase

Polymerase chain reaction (PCR) was performed with two polymerases. Thermus aquaticus DNA polymerase (Taq), and modified T7 DNA polymerase (Sequenase). Both polymerases were used to amplify the same portion of the human 18S rRNA gene. We report a PCR artifact, namely a deletion of 54 bp, when Taq polymerase was used to amplify a portion of the human 18S rRNA gene. PCR performed with Sequenase did not produce this artifact. The deletion eliminated a potentially stable hairpin loop. Our data are consistent with the following model for generation of the deletion: (i) the formation of an intrastrand hairpin, and (ii) polymerization across the base of the hairpin, thus deleting the nucleotide sequence in the hairpin. Furthermore, we show that the deletion occurs mainly during synthesis of the (-)DNA strand. Our observations suggest that similar artifacts may occur in other sequences containing stable secondary structures.     

Development of a new DNA sequencing method: 3'-ester cleavage catalyzed by Taq DNA polymerase.

A new 3'-esterified dTTP is incorporated into DNA by Taq DNA polymerase but does not act as a chain terminator. The esterase activity of the polymerase seems to be template dependent and occurs only if the next correct nucleotide is present.