Characterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter species

The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 degrees C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile.     

Detection of subpicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor

We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20nm and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1pM MMP-2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations.     

Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombination between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.     

Differential magnification of rDNA gene types in bobbed mutants of Drosophila melanogaster

Summary In Drosophila melanogaster a partial loss of ribosomal genes leads to the bobbed phenotype. Magnification is a heritable increase in rDNA that may occur in males carrying a deleted X chromosome with a strong bobbed phenotype. The restriction patterns of X chromosome total rDNA, insertions and spacers from magnified bobbed strains were compared with those of the original bobbed mutations. It was found that magnification modifies restriction patterns and differentially affects gene types, increasing specific genes lacking insertions (INS^-). Increases in copy number of genes with type I insertions are generally lower than the total number of INS^- genes, while type II insertion genes are not perceptibly increased. The recovery of homogeneous progeny from a single premagnified male indicates that the magnification event might take place and become stable very early in the germ line, arguing against magnification being due to extrachromosomal amplification. Additionally, some gene types increase 3.5-fold while others are eliminated, indicating that they could not result from a single unequal cross-over. These results are in good agreement with the existence of partial clustering of rDNA genes according to type, and suggest that magnification could result from local amplification of genes.     

扩增条件对茶类植物RAPD带的影响

采用梯度分析的方法试验了模板ＤＮＡ、引物、镁离子、ｄＮＴＰ和Ｔａｑ酶的浓度对茶类植物进行ＲＡＰＤ分析中ＤＮＡ扩增结果的影响。实验表明这些条件的变化对扩增出来的ＲＡＰＤ带的数目和强弱会产生影响。经过比较分析,筛选出对于茶类植物进行ＲＡＰＤ分析较理想的扩增条件：２．０ｍｍｏｌ／ＬＭｇＣｌ２,２００ｕｍｏｌ／ＬｄＮＴＰ,１５ｎｇ引物／２０ｕｌ反应体积,４ｎｇ模板ＤＮＡ／ｕｌ反应体积,１ＵＴａｑ酶／２０ｕｌ反应体积。     

孓遗植物桫椤RAPD分析中主要反应参数的影响

为得到桫椤RAPD扩增的最佳条件,对影响桫椤RAPD扩增的模板纯化方法、模板含量、酶浓度、Mg²⁺浓度、dNTP浓度和引物浓度及扩增程序等多种因素进行了探讨.结果表明,用玻璃奶和蛋白酶K纯化后获得的DNA作为模板进行RAPD反应,其扩增带比用未纯化的和仅用无水乙醇再沉淀的DNA为模板更亮,更清晰,重复性更好;模板浓度、酶浓度、Mg²⁺及dNIT浓度、引物浓度及退火温度这些因素都会对RAPD产生影响.经过本实验的探索,发现桫椤RAPD扩增的最佳体系是(2.5×10^-2mL反应体积):模板浓度为70ng,Mg²⁺浓度为2.5mmol·L^-1,dNTP为0.2mmol·L^-1,引物3×10^-4mL;最佳扩增程序为:94℃200s,一个循环;94℃60s,36℃60s,72℃120s,40个循环:72℃600s,一个循环:4℃保存.     

Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64-99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.     

灯盏花 chi 的克隆及其生物信息学分析

查尔酮异构酶（CHI）是调控黄酮生物合成的关键酶,分离和克隆这一酶的功能基因,对利用转基因技术进行灯盏花黄酮生物合成的调控具有重要意义。本研究采用RT-PCR和RACE技术,获得了chi cDNA全序列,GenBank登录号为GU208823.1,序列全长996 bp,开放阅读框为594 bp,编码197个氨基酸,3-Race有一个多聚腺苷酸加尾信号。应用软件预测该基因编码蛋白分子量约为21.6 kD,理论等电点为4.78。该基因编码的蛋白无跨膜结构域,其二级结构的主要构件为α-螺旋和随机卷曲。对其三级结构进行了建模,表明其结构与苜蓿chi的三级结构相似。同时根据灯盏花chi N端序列变化的特征,提出了灯盏乙素的合成可能与chi在细胞亚结构的定位及其与合成代谢相关酶形成复合酶的特异性有关。研究为利用基因工程定向改变灯盏花黄酮代谢产物奠定了基础。     

Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25 min at 63 °C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83 bp and 89 bp sized bands and the LAMP product melt curves showed consistent melting temperature (T_m) of ∼89 °C. The assay analytical sensitivity is ∼0.1 tryps/ml while that of classical PCR test targeting the same gene is ∼10 tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.