Development of a Novel Electrochemical Biosensor for Detection and Discrimination of DNA Sequence and Single Base Mutation in dsDNA Samples Based on PNA‐dsDNA Hybridization – a new Platform Technology

In spite of the extensive attention paid on the development of various DNA detection strategies, very few studies have been reported regarding direct detection of DNA sequence and mutation in dsDNA. Here, we describe the feasibility of detection and discrimination of target DNA sequences and single base mutations (SBM) directly in double‐stranded oligonucleotides and PCR products without the need for denaturation of the target dsDNA samples. This goal was achieved by employing a peptide nucleic acid (PNA) chain, self‐assembled on the gold electrode as a probe, which binds to dsDNA and forms PNA‐dsDNA hybrid.     

Application of Legendre Polynomials Correction Before Component Quantification of Biological Mid-infrared Spectra

Abstract

Quantitative determination from infrared spectra still faces problems that are not completely resolved to date. One of these problems is the deformation of baselines.

We have tested the Legendre baseline correction method by subtracting from a spectrum the low-degreed terms from a Legendre polynomial function which is obtained by the decomposition of the spectrum.

As opposed to the filtered spectra with n=0 which remained very similar to the original spectra, the filtered spectra, as from n=2, exhibit a great amount of distorsion. This result suggests that it is as from n=2, that the terms that are meaningful to the signal (not just to baseline), are to be substracted.

Mathematical methods such as Principal Component Analysis and Principal Component Regression are used to analyse quantitatively components from biological samples. The thus predicted sucrose content values for values of n that range between 0 and 8 (n={0, 2, 4, 6, 8}) show that the best results are obtained for n=4. The mean and standard deviation values of the difference between reference and predicted values varies between 8.5 × 10^?2 and 6.6 × 10^?2 and between 0. 224 and 0. 195 respectively; these values are minimum when the n=4 term is subtracted from the spectra.

This processing hence, sensibly improves the quantification of components from Mid-FTIR spectra of biological solutions. This procedure would be interesting in cases where the precision of quantitative measures is essential.     

D38R突变对细菌视紫红质光循环后半程M态和N态的影响

用重叠PCR的方法，定点突变细菌视紫红质（bacteriorhodopsin，BR），克隆到单突变体BRD96N和双突变体BRD38R/D96N的基因，同源转化盐沼盐杆菌（Halobacterium salinarium）L33（BR），表达突变蛋白BRD96N和BRD38R／D96N通过显微视频方法对突变BR的M态光致变色特性进行记录，BRD38R／D96N的M态寿命为5s，约为BRD96N（M态寿命为3s）的1．7倍．对突变体的激光共聚焦拉曼光谱测定发现，在BRD96N中，1170cm^-1下移到1171cm^-1，1258cm^-1上移到1255cm^-1，而BRD38R/D96N中1170cm^-1下移到1173cm^-1；1258cm^-1。上移到1252cm^-1，而且BRD38R/D96N与BRD96N相比，1186cm^-1处的峰明显增强．近紫外区圆二色谱显示，两种突变体近紫外区的正负带虽然没有太大差别，但288nm和290nm处的极峰却差别显著．所有这些研究结果表明，细菌视紫红质Asp38（D38）突变为Arg（R）可以延长其光循环后半程M态和N态的寿命．     

中国人CD59cDNA克隆、序列分析及其表达

用ＲＴ－ＰＣＲ方法从中国人胚胎总ｍＲＮＡ中扩增出４２０ｂｐ人补体终末阶段同源限制因子（ＣＤ５９）的ｃＤＮＡ，序列分析结果表明，所获得的ＣＤ５９ｃＤＮＡ与文献报道中的基因序列完全一致，含ＣＤ５９全长编码区．构建了真核表达质粒，在３Ｔ３细胞中得到了表达．     

无胶毛细管电泳分离碱基对范围宽的DNA片段

提出一种无胶毛细管电泳分离碱基对范围宽的ＤＮＡ片段的方法．用ＤＢ－１气相色谱毛细管柱，以羟乙基纤维素为筛分介质，研究了λＤＮＡ／ＥｃｏＲⅠ＋ＨｉｎｄⅢ限制性片段的分离，分离效率达１．２×１０６板／ｍ，检测限为２．１×１０－１７ｍｏｌ．应用于一种鲤鱼种族鉴别基因的聚合酶链反应（ＰＣＲ）扩增产物的分离鉴定，结果与实际相符，分离速度比传统电泳方法提高２０倍．     

相位共轭光腔的热稳性

使用传播矩阵方法推导出了相位共轭腔的热稳条件,指出仅满足G_1G_2=(1/2)并不意味着相位共轭腔一定是热稳的.因此,为得到高质量的输出激光束,需进行相位共轭腔的最佳设计.用本文所得结果还能解释利用相位共轭技术消除畸变的定域性现象.     

Analysis of electrophoretic patterns of arbitrarily primed PCR profiling

We present a mathematical algorithm for the analysis of electrophoretic patterns resulting from arbitrarily primed PCR profiling. The algorithm is based on the established mathematical procedures applied to the analysis of digital images of gel patterns. The algorithm includes (a) transformation of the image into a matrix form, (b) identification of every electrophoretic lane as a set of matrix columns that are further mathematically processed, (c) averaging of matrix columns corresponding to electrophoretic lanes that define lane representatives, (d) elimination of "smiling" bands, (e) solving the problem of a lane offset, and (f) removal of the background. Representation of individual electrophoretic lanes in the form of functions allows interlane comparisons and further mathematical analysis. Direct comparison of selected lanes was obtained by employing correlation analysis. Gel images were those obtained after arbitrarily primed PCR analysis of DNA that underwent damage induced by gamma radiation from a (60)Co source. The applied method proved to be useful for elimination of subjectivity of visual inspection. It offers the possibility to avoid overlooking important differences in case of suboptimal electrophoretic resolution. In addition, higher precision is achieved in the assessment of quantitative differences due to better insight into experimental artifacts. These simple mathematical methods offer an open-type algorithm, i.e., this algorithm enables easy implementation of different parameters that may be useful for other analytical needs.     

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.     

Helicobacter pylori determination in non-municipal drinking water and epidemiological findings

Background.?Studies conducted in Europe as well as in North and South America have tried to link Helicobacter pylori colonization with the drinking water supply, especially since H. pylori is known to survive quite well in water.

Methods.?In 2000, a cohort of 1884 grade-two children from two rural counties surrounding the city of Leipzig, Germany (77.4% of the 1991/1992 birth cohort) were tested for H. pylori colonization using the [¹³C]urea breath test. A parent-completed questionnaire elicited details on living conditions and lifestyle habits including questions on the children's drinking water from sources other than public water supplies, swimming in natural waters, etc.

In a second independent study, samples of well water, taken from 157 private wells still used in the two counties, were being tested for the presence of H. pylori, using polymerase chain reaction (PCR) method to determine relevant target DNA fragments of H. pylori.

Results.?In county I, 5.7% of the children and in county II 6.6% tested H. pylori-positive. Cluster analyses of the questionnaire data in both counties pointed to ‘drinking water from other than municipal sources’, as the closest H. pylori-associated cluster variable. The cluster estimations were supported by odds ratio (OR) calculations with an OR?=?16.4 (95% confidence interval (CI) 3.1,…,88.5) for county I and OR?=?4.0 (95% CI 1.3,…,12.4) for county II.

The PCR analyses showed H. pylori DNA fragments in 10.8% of the wells in county I and 9.2% in county II. The detection limit was set at 10 DNA copies corresponding to 125?bacteria/L, the average infestation of these wells was 931?bacteria/L.

Conclusion.?Despite the fact that the microbiological and epidemiological data do not correspond except that both studies were conducted in the same geographical areas, the independent findings of H. pylori in well water in the same general areas where children do seem to drink water other than from the public water supply suggests that water may be an important source of H. pylori infection.     

Modeling based on subspace orthogonal projections for QSAR and QSPR research

A novel projection modeling method for quantitative structure activity relationship (QSAR) and quantitative structure property relationship (QSPR) is developed in this paper. Orthogonalization of block variables is introduced to deal with the problem of variable selection. Projections based on least squares are used to construct the modeling space in order to search for the best regression directions for chemical modeling. A suitable prediction space for such a model is further defined to confine the usage range of the model. Three real data sets were analyzed to check the performance of the proposed modeling method. The results obtained from Monte‐Carlo cross‐validation (MCCV) showed that the proposed modeling method might provide better results for QSAR and QSPR modeling than PCR and PLS with respect to both fitting and prediction abilities. Copyright © 2007 John Wiley & Sons, Ltd.