New specificity and yield enhancer of polymerase chain reactions

Tetramethylammonium (TMA) chloride, dimethyl sulfoxide and formamide are known to increase, under certain conditions, the specificity and efficiency of the polymerase chain reaction (PCR). We compared the ability of several TMA derivatives and some other reagents to increase the specificity of PCR and to improve the yield of amplification. A novel combination of the enhancer TMA and oxalate as anion is demonstrated to be a powerful enhancer of PCR. Addition of 2 mM TMA oxalate to the PCR mixture decreases the formation of non-specific DNA fragments and increases the yield of specific PCR products.     

PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).     

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.     

Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B

The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo–) and Deep Vent (exo–) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chromophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.     

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.     

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.     

Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA

PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.     

多倍体罗汉果PCR-RFLP参数的优化与应用

以多倍体罗汉果DNA为材料,采用L16(4~5)正交组合试验和单因素梯度试验,研究Mg~(2+)、dNTP、引物、Taq DNA聚合酶、模板DNA浓度和退火温度、循环次数等对PCR扩增结果以及内切酶量、酶切时间对酶切反应的影响。结果表明,多倍体罗汉果RFLP最优PCR反应体系和扩增参数为:在25μL扩增反应体系中,10×Buffer 2.5μL,MgCl_2 1.5 mmol/L,dNTP 0.2 mmol/L,引物0.1μmol/L,Taq DNA聚合酶2.0 U,模板DNA 60 ng;退火温度为56℃,循环次数为35次。酶切反应体系:内切酶10×Buffer 2.0μL,内切酶5.0U,PCR产物15μL,超纯水补至20μL;酶切时间2 h。     

线虫PCR:直接针对秀丽线虫进行PCR扩增基因组DNA的新方法

目的:直接针对秀丽线虫进行PCR反应,以便快速扩增基因组DNA,从而提高钓取目的基因和鉴定基因组是否发生突变的效率.方法:根据生物信息学分析,针对不同基因设计单重或多重PCR引物;在不含砌DNA聚合酶的PCR反应体系中加入蛋白酶K消化秀丽线虫染色体中的组蛋白,然后加入Taq酶,直接针对野生型或突变型秀丽线虫个体进行PC...     

木麻黄SSR-PCR反应体系的建立与优化

为得到木麻黄SSR-PCR反应的最佳体系,以4个种(短枝木麻黄、山地木麻黄、粗枝木麻黄、细枝木麻黄)的24个无性系为材料,采用L₁₆(4⁵)正交设计对影响木麻黄SSR-PCR反应的4个因素(Taq酶、dNTP、Mg²⁺和引物)在4个水平上进行了优化,并利用直观分析和方差分析两种方法对PCR结果进行评价。结果表明:引物、Mg²⁺和dNTP均对木麻黄SSR-PCR反应结果有极显著的影响(P<0.01),影响程度由大到小为:引物>Mg²⁺>dNTP,而Taq酶对扩增结果无显著影响;确定了两种木麻黄SSR-PCR反应体系(体系6和体系15),体系6为1×PCR buffer、2ng模板DNA、0.5μmol·L^-1引物、1.5 mmol·L^-1 Mg²⁺、0.1 mmol·L^-1 dNTP、0.5 U Taq酶;体系15为1×PCR buffer、2ng模板DNA、0.5μmol·L^-1引物、1.75 mmol·L^-1 Mg²⁺、0.2 mmol·L^-1 dNTP、1.25 U Taq酶,反应体系共10μL,不足部分用ddH2O补足。从节约成本和降低非特异性产物的角度考虑可将体系6作为最佳体系,体系15为备用体系;本试验所选荧光引物M26、M36的退火温度在52~62℃均可扩增出清晰明亮的条带。为简化操作步骤和减少非特异性产物,可选择60℃作为引物M26、M36的最佳退火温度。     

Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5, 6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage lambda DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.     

丹参ISSR-PCR反应体系的建立与正交优化

利用正交试验设计的方法,从引物浓度、Taq DNA聚合酶浓度、Mg2+浓度、dNTP浓度4种因素3个水平,对丹参ISSR-PCR反应体系进行优化分析,并在此基础上对模板DNA浓度、PCR反应过程中的退火温度进行梯度检测。结果表明:20μL ISSR-PCR反应体系中各因素的最佳浓度为1×PCR buffer、200μmol/L dNTP、1.0μmol/L引物、1.5mmol/L Mg2+和1 U Taq DNA聚合酶,最佳模板DNA浓度为20～60ng,引物UBC 835的最佳退火温度为51.7℃。     

CYP2C19基因多态性PCR扩增体系的正交优化与验证

本实验通过建立CYP2C19*2、*3和*17基因多态性PCR反应体系和条件,筛选出4μL体系中各因素最佳水平。采用SNaPshot技术对CYP2C19基因3个SNP位点*2、*3和*17同时进行复合扩增检测,利用L9(34)正交实验设计,对影响PCR反应体系和条件的3个因素(PCR Mix, Taq DNA聚合酶,循环次数)在3个水平上进行优化,结果采用综合评分法和极差分析法进行分析。用3组已知样本对正交优化所得条件进行重复性和稳定性验证。结果表明CYP2C19*2、*3和*17基因PCR扩增体系的影响因素依次为:PCR Mix>循环数>Taq DNA聚合酶。最佳反应体系为PCR Mix 2.0μL、Taq DNA聚合酶0.2μL、循环次数32次。3组样本验证效果满意。优化的CYP2C19*2、*3和*17基因PCR反应体系稳定性高,重复性和经济性较好,为该基因多态性的大规模调查奠定了基础。     

High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.

A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.     

黄皮SRAP反应体系优化正交实验研究

以黄皮(Clausena lansium)‘甜黄皮’品种为试材,利用正交设计L₁₆(4⁵)对黄皮SRAP-PCR反应体系中的5因素(Taq聚合酶、Mg²⁺、模板DNA、dNTPs、引物)在4个水平上进行优化试验。结果表明,不同因素对黄皮SRAP反应体系影响从大到小的顺序为:Mg²⁺和Taq聚合酶> 模板DNA> 引物> dNTPs;初步确立了适合黄皮的SRAP-PCR扩增体系为:在25 μl反应体系中,包括10×PCR buffer 2.5 μl、Taq DNA聚合酶0.75U、Mg²⁺ 2.0 mmol/L、模板DNA 60 ng、dNTPs 0.2 mmol/L、引物0.2 μmol/L。     

Respiratory chain inhibition by fullerene derivatives: hydrogen peroxide production caused by fullerene derivatives and a respiratory chain system

Fullerene is a new type of carbon allotrope. We have shown that the fullerene derivative C(60)-bis(N,N-dimethylpyrrolidinium iodide), a regio isomer mixture, inhibited Escherichia coli growth and dioxygen uptake caused by E. coli and glucose. This result indicates that the mechanism of the bacteriostatic effect is the inhibition of energy metabolism. In this study, we isolated two regio isomers of C(60)-bis(N,N-dimethylpyrrolidinium iodide) and studied their effect on E. coli growth and on respiratory chain activity. In dioxygen uptake caused by the inner-membrane and NADH, the effect of fullerene derivatives was biphasic. At low concentrations of both fullerene derivatives, dioxygen uptake was inhibited, whereas at high concentrations, it was increased. At high concentrations, consumed dioxygen was converted to H(2)O(2). An electrochemical study revealed that reduced fullerene derivatives react with dioxygen. This activity was closely related to a redox property of the isomers.     

A quick and inexpensive method for removing polysaccharides from plant genomic DNA.

A quick and inexpensive method has been demonstrated to remove polysaccharide contamination from plant DNA. Isolated plant genomic DNA with polysaccharide contaminants was dissolved in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) with NaCl ranging from 0.5-3.0 M, then precipitated with two volumes of ethanol. Most of the polysaccharides were removed effectively in a single high-salt precipitation at 1.0-2.5 M NaCl. At 3.0 M NaCl, the salt precipitated out of solution. Purified DNA was easily digested by either HindIII or EcoRI and was satisfactory as a template for PCR. The results show that high-salt precipitation effectively removed polysaccharides and their inhibitory effects on restriction enzyme and Taq polymerase activity.     

Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10–100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1–1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.     

怀地黄ISSR扩增条件优化的研究

用CTAB法提取怀地黄嫩叶DNA,进行简单重复间序列标记(ISSR)分析.通过单因子实验分别研究了退火温度、Taq酶单位、Mg2+浓度、dNTP浓度、引物浓度和模板DNA浓度对ISSR-PCR反应的影响,找出各自的合适条件,而且每一个合适条件确定以后都被作为后续研究的一个条件.通过各个因子的组合研究建立了适宜于怀地黄ISSR分析的扩增体系25 μL PCR反应体积,1×Taq DNA酶缓冲液(10 mmol/L Tris-HCl,50 mmol/L KCl,0.1% Trion X-100,pH9.0 ),2.5 mmol/L MgCl2,1.5～1.0 U Taq酶,60 ng模板DNA,0.4 μmmol/L引物,各0.4 mmol/L的dATP、dGTP、dCTP和dTTP.合适的退火温度为53～55℃.为用ISSR技术分析鉴定怀地黄种质资源奠定了良好的基础.