A simple and eficient method for high fidelity PCR cloning using antibody-neutralizing technology

We introduce the TA cloning antibody method for the high-fidelity PCR product amplified by family B DNA polymerase without purification. This method uses antibodies and Thermus aquaticus (Taq) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning efficiency to that of the PCR product of Taq.     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 “inhibited” crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.     

Cloning,expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, <Emphasis Type="Italic">Thermococcus celer</Emphasis>

The family B DNA polymerase gene was amplified from Thermococcus celer genomic DNA by using the degenerate primers and DNA walking PCR. The Tce DNA polymerase gene was cloned and sequenced. The gene contains an ORF of 2,325 bp encoding 774 amino acid residues with a calculated molecular weight of 89,788.9 kDa. The Tce DNA polymerase was purified by heat treatment and heparin column chromatography. The optimal conditions for PCR were determined. Long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tce DNA polymerases (Tce plus DNA polymerase). Tce plus DNA polymerase surpassed the PCR performance of Tce, Taq and Pfu DNA polymerases in terms of yield and efficiency.     

Efficient molecular cloning of environmental DNA from geothermal sediments

An efficient and simple method for constructing an environmental library using mechanically sheared DNA obtained directly from geothermal sediments is presented. The method is based on blunt-end modification of DNA fragments followed by 3-adenylation using Vent DNA polymerase and Taq DNA polymerase, respectively. The prepared DNA fragments are then ligated into a TA cloning vector and used in the transformation of Escherichia coli. This method has been successfully applied to the cloning of ORFs derived from uncultivated prokaryotes present in geothermal sediment.     

Influence of DNA aptamer structure on the specificity of binding to Taq DNA polymerase

The secondary structure of DNA aptamer to Taq DNA polymerase was established as a hairpin. Both stem and loop structures of DNA ligand were shown to be involved in the interaction with Taq DNA polymerase. Moreover, the structure and sequence of DNA aptamer that was the most effective inhibitor of DNA polymerase activity were established. This crucial structure was evaluated as a GC-rich stem longer than 17 bp, and a loop consisting of 12 bases with strictly determined nucleotide sequence. It was demonstrated that nucleotide in position 23 counting from the 5"-end of DNA ligand was involved in direct contact with Taq DNA polymerase. The ability of optimized DNA aptamer TQ21-11 to form a complex with the enzyme was increased 5-fold in comparison to the initial aptamer.     

Incorporation of Multiple Sequential Pseudothymidines by DNA Polymerases and Their Impact on DNA Duplex Structure

Thermal denaturation and circular dichroism studies suggested that multiple (up to 12), sequential pseudothymidines, a representative C-glycoside, do not perturb the structure of a representative DNA duplex. Further, various Family A and B DNA polymerases were found to extend a primer by incorporating four sequential pseudothymidine triphosphates, and then continue the extension to generate full-length product. Detailed studies showed that Taq polymerase incorporated up to five sequential C-glycosides, but not more. These results constrain architectures for sequencing, quantitating, and analyzing DNA analogs that exploit C-glycosides, and define better the challenge of creating a synthetic biology using these with natural polymerases.     

Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase

The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 × 10⁻⁶) than Taq DNA polymerase (11.98 × 10⁻⁶). Uniquely, Neq DNA polymerase also amplified λ DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.     

Improved thermostability and PCR efficiency of Thermococcus celericrescens DNA polymerase via site-directed mutagenesis

The Thermococcus celericrescens (Tcel) DNA polymerase gene, which contains a 2328-bp open reading frame that encodes 775 amino acid residues, was expressed in the Escherichia coli strain Rosetta(DE3)pLysS. The expressed enzyme was purified through heat treatment, HisTrap™ HP column chromatography and then HiTrap™ SP HP column chromatography. Tcel DNA polymerase has poor thermostability and PCR efficiency compared to those of other family B DNA polymerases. To improve thermostability and PCR efficiency, mutant Tcel DNA polymerases were created via site-directed mutagenesis. Specifically, we targeted the A752 residue for enhanced thermostability and the N213 residue for improved PCR efficiency. The mutant Tcel DNA polymerases all showed enhanced PCR efficiency and thermostability compared to those of the wild-type Tcel DNA polymerase. Specifically, the double mutant TcelA752K/N213D DNA polymerase had an approximately three-fold increase in thermostability over that of the wild-type enzyme and amplified a long 10-kb PCR product in an extension time of 2 min. However, there was a small change in the 3′ → 5′ exonuclease activity compared with that of the wild-type Tcel DNA polymerase, even though the mutation is in the ExoII motif. The double mutant TcelA752K/N213D DNA polymerase had a 2.6-fold lower error rate compared to that of Taq DNA polymerase. It seems that the double mutant TcelA752K/N213D DNA polymerase can be used in LA (long and accurate) PCR.     

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap™ Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris–HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH₄)₂SO_4, 2 mM MgCl_2, 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).     

Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of ω-functionalized thiols

A new efficient immobilization method that enables oriented immobilization of biologically active proteins was developed based on concepts of active site masking and kinetic control. Taq DNA polymerase was immobilized covalently on mixed self-assembled monolayers (SAMs) of ω-carboxylated thiol and ω-hydroxylated thiol through amide bonds between the protein and the carboxyl group in SAMs. Activity of the immobilized enzyme as large as 70% of solution-phase enzyme was achieved by masking the active site of the Taq DNA polymerase prior to the immobilization. In addition, the number of immobilization bonds was controlled by optimizing the carboxyl group concentration in the mixed monolayer. The maximum activity of immobilized Taq DNA polymerase was achieved at 5% of 12-mercaptododecanoic acid. The activity observed with protected immobilized enzyme was approximately 20 times higher than that observed with randomly immobilized enzyme. The maximum activity was acquired at a 1:1 DNA/enzyme masking ratio, immobilization pH 8.3, and within 10 min of reaction time. This concept of the active site masking and kinetic control of the number of covalent bonds between proteins and the surface can be generally applicable to a broad range of proteins to be immobilized on the solid surface with higher activity.     

New DNA polymerase from the hyperthermophilic marine archaeon <Emphasis Type="Italic">Thermococcus thioreducens</Emphasis>

The family B DNA polymerase gene of Thermococcus thioreducens, an archaeon recently isolated from the Rainbow hydrothermal vent field, was cloned and its protein product expressed, purified and characterized. The gene was found to encode a 1,311 amino acid chain including an intein sequence of 537 residues. Phylogenetic analysis revealed a predominantly vertical type of inheritance of the intein in the Thermococcales order. Primary sequence analysis of the mature protein (TthiPolB) showed significant sequence conservation among DNA polymerases in this family. The structural fold of TthiPolB was predicted against the known crystallographic structure of a family B DNA polymerase from Thermococcus gorgonarius, allowing regional domain assignments within the TthiPolB sequence. The recombinant TthiPolB was overexpressed in Escherichia coli and purified for biochemical characterization. Compared with other DNA polymerases from the Thermococcales order, TthiPolB was found to have moderate thermal stability and fidelity, and a high extension rate, consistent with an extremely low K _m corresponding to the dNTP substrate. TthiPolB performed remarkably well in a wide range of PCR conditions, being faster, more stable and more accurate than many commonly used enzymes.     

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. Revisions requested 1 November 2005; Revisions received 28 November 2005     

Optimization of PCR in application of hot start <Emphasis Type="Italic">Taq</Emphasis> DNA polymerase for detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> with primers FER1-F and FER1-R1

There are two approaches in detection of bacterium Erwinia amylovora by PCR. One is based on detection of plasmid pEA29 and the other is based on detection of a chromosomal DNA sequence, specific for E. amylovora, in a sample. Since pathogenic strains without pEA29 have been isolated from the environment, methods based on this plasmid have been compromised and PCR methods based on chromosomal DNA species specific sequences became only reliable methods. PCR method with chromosomal primers FER1-F and FER1-R is currently the most reliable method due to its high sensitivity and specificity. The goal of this research is to make a significant improvement of the method by optimization of PCR in application of hot start DNA Taq polymerase, instead of wax, to obtain a hot start reaction. This enzyme, which is currently widely applied, can provide simpler achievement of hot start, saving labor and time and decreasing possibility of cross contamination of samples. Experiments showed that simple replacement of a regular recombinant Taq DNA polymerase by a hot start Taq DNA polymerase leads to complete failure of the reaction. Many optimization experiments had to be carried out to obtain an operational and reliable PCR which simultaneously has high sensitivity and specificity. Content of the reaction mixture, as well as temperature and time parameters of PCR, were significantly changed to achieve proper optimization.     

New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals

This work aims to study molecular mechanisms involved in the formation of DNA-containing microparticles and nanoparticles during PCR. Both pyrophosphate and Mg²⁺ ions proved to play an important role in the generation of DNA microparticles (MPs) with a unique and sophisticated structure in PCR with Taq polymerase. Thus, the addition of Tli thermostable pyrophosphatase to a PCR mixture inhibited this process and caused the destruction of synthesized DNA MPs. Thermal cycling of Na-pyrophosphate (Na-PPi)- and Mg²⁺-containing mixtures (without DNA polymerase and dNTPs) under the standard PCR regime yielded crystalline oval or lenticular microdisks and 3D MPs composed from magnesium pyrophosphate (Mg-PPi). As shown by scanning electron microscopy (SEM), the produced Mg-PPi microparticles consisted of intersecting disks or their segments. They were morphologically similar but simpler than DNA-containing MPs generated in PCR. The incorporation of dNTPs, primers, or dsDNA into Mg-pyrophosphate particles resulted in the structural diversification of 3D microparticles. Thus, the unusual and complex structure of DNA MPs generated in PCR is governed by the unique feature of Mg-pyrophosphate to form supramolecular particles during thermal cycling. We hypothesize the Mg-pyrophosphate particles that are produced during thermal cycling serve as scaffolds for amplicon DNA condensation.     

Characterization and application of aptamers for Taq DNA polymerase selected using an evolution-mimicking algorithm

Using an evolution-mimicking algorithm (EMA), we have recently identified DNA aptamers that inhibit Taq DNA polymerase. In the present study, we have attempted to improve further the inhibitory activities of aptamers, as well as to characterize those aptamers with the most potent inhibitory activities. To characterize the most potent aptamer and demonstrate its applicability, the abilities to inhibit Tth DNA polymerase and to modulate specific amplification in PCR were investigated. This aptamer inhibited both Tth DNA polymerase and Taq DNA polymerase and improved the specificity of detection of a low-copy-number target gene in PCR using these DNA polymerases.     

Characteristics of microspheres formed in PCR with bacterial genomic DNA or plasmid DNA as templates

Earlier, we discovered that, along with linear DNA fragments, nano- and microparticles of DNA and their aggregates are formed in the PCR with yeast genomic DNA used as a template and gene-specific or partially complementary primers. The size of the microparticles (microspheres) varied in the range of 0.5 to 3–4 μm. Only thermostable KlenTaq polymerase but not Taq polymerase could effectively generate microspheres. In this work, we demonstrate that KlenTaq polymerase can produce microspheres of variable size (1 to 7 μm in diameter) if genomic DNA of the bacterium Acidithiobacillus ferrooxidans and partially complementary primers are present in the PCR mixture. Conditions for generation of DNA microparticles in PCR with Taq-polymerase and bacterial genomic DNA as template were also elaborated. It was also found that mainly large microspheres of up to 7 μm accumulated in PCR with plasmid DNAs used as templates and gene-specific primers in the presence of KlenTaq polymerase or mixtures of KlenTaq and Pfu polymerases. Besides, small aggregates, as well as linear branched structures and three-dimensional conglomerates of fused microspheres, were also revealed in the PCR mixtures. UV absorption spectra of native DNA microspheres and microspheres that had undergone heating at 93°C were registered. The key role of Mg²⁺ cations in the formation and stabilization of the microsphere structure was established.     

News & Notes: Efficient Library Construction with a TA Vector and Its Application to Cloning of the Phytoene Synthase Gene from the Cyanobacterium Spirulina platensis

An efficient and simple method for constructing a genomic DNA library is presented by use of a TA cloning vector. It is based on sonicative cleavage of genomic DNA and modification of the fragment ends with Taq DNA polymerase, followed by ligation with a TA vector. This method was successfully applied to cloning of the phytoene synthase gene crtB from Spirulina platensis. The method is useful when the genomic DNA is not well digested with restriction enzymes owing to methylation or other reasons. Received: 25 February 1988/Accepted: 12 May 1998     

Micro- and nanoparticles of condensed DNA Formed in PCR with <Emphasis Type="Italic">Taq</Emphasis> polymerase and plasmid DNA as a template

Formation of micro- and nanoparticles of condensed DNA during PCR with microbial genomic DNA or plasmid DNA as templates was reported previously. Initially, the microparticles were formed using a thermostable KlenTaq polymerase, which is a deletion variant of Taq polymerase. The present work shows that Taq polymerase is also capable of efficient formation of micro- and nanoparticles of condensed DNA in PCR. Electron microscopy revealed a number of morphological types (more than four) of microparticles produced in PCR with different reaction buffers in the presence of Taq polymerase and different plasmid DNAs as a template. In the case of some kinds of amplicons, an increase in the number of thermal cycles was shown to result in production of numerous nanowires and electron-dense spherical nanoparticles. The PCR conditions for preferential formation of discs (or ellipsoids) a few micrometers in diameter and several dozens of nanometers in thickness were determined. The structure of microparticles formed in the presence of Taq polymerase was found to depend on the level of synthesis of single-stranded DNA fragments in PCR. Experiments with nuclease S1 revealed that, along with double-stranded DNAs of the amplicon, micro- and nano-particles contained single-stranded DNA fragments, which were absolutely necessary for their formation. In light of these data, the molecular mechanism of micro- and nanoparticle formation in the course of PCR is discussed.     

Simplified DNA Extraction and Improved PCR Based Detection of Greening Bacterium in Citrus

Citrus greening disease caused by a fastidious bacterium is an important graft transmissible disease in commercial citrus in India and other parts of the world. Polymerase chain reaction (PCR) is a sensitive and convenient method for detection of greening bacterium. A non-phenol chloroform method of DNA extraction was evaluated for DNA quality and PCR based detection of greening bacterium. The method was comparable with a commercial DNA extraction kit (Qiagen) and better than a CTAB based DNA extraction method. To improve the reliability, three primer sets (primers A, B, and C yielding amplicons of 1160 bp, 703 bp and 451 bp, respectively) and two polymerase enzymes (Taq polymerase and Klen Taq polymerase) were evaluated. The primer set C provided better amplification when compared to primer sets A and B. Primer C in combination with Taq polymerase provided amplification band at a DNA template concentration of 100 pg but good amplification band was obtained at still lower DNA template concentration of 0.1 pg when Klen Taq polymerase was used. The standardized PCR protocol combining non-phenol chloroform method of DNA isolation, primer set C and Klen Taq polymerase enzyme was found very effective in detecting greening bacterium in citrus trees. The sequence of cloned amplicon from 16S ribosomal RNA gene had 89–100 % sequence identity with corresponding sequence of Candidatus Liberibacter asiaticus from China, Brazil, Japan and Pune isolate of India, C. Liberibacter americnus from Brazil and C. Liberibacter africanus from Africa.