Three-step PCR mutagenesis for 'linker scanning'.

'Linker scanning' has been used as an efficient method for systematically surveying a segment of DNA for functional elements by mutagenesis. A three-step PCR method was developed to simplify this process. In this method, a set of 'mutation primers' was made with 6 to 8 base substitutions in the center of the primers. In the first PCR reaction, these 'mutation primers' are paired with an 3' primer from the opposite end of the analyzed sequences to form a 'ladder' of fragments containing the base pair substitutions. These are used as templates in the second PCR with the 3' primer as the only primer to generate single stranded sequences, which are used as primers in the third PCR paired with an 5' primer to complete the mutagenesis. We have tested the method in a mutation screen of the steroid sulfatase promoter. Its application to general site specific mutagenesis is discussed.     

A likelihood approach for functional response models

Functional response is an important determinant of community dynamics, and thus empirical methods for characterizing functional responses are as important in understanding ecological processes. The most commonly used method is based on the sum of squares, and the maximum likelihood method is rarely used. When the likelihood method is used, potentially inappropriate probability distributions such as binomial distributions are typically assumed for the number of prey eaten in experiments. In this study, I present a likelihood approach in which the probability distributions are generated by mechanistic understanding of predation processes using Monte Carlo simulations. An example is given on the Holling type II functional response model, but the method is flexible and allows characterization of a wide variety of functional response models. In the example, the likelihood method consistently resulted in superior estimates than the least squares method.     

蚜虫中携带马铃薯卷叶病毒检测方法的改进

马铃薯卷叶病毒( Potato leafroll virus,PLRV)对马铃薯生产的危害极大,是一种极为重要的马铃薯病毒病。 RT－PCR是马铃薯卷叶病毒检测较为常用的方法,该方法检测准确率高、成本低、适用范围广。但在实际生产中其检测对象多为染病植株,对PLRV传播的主要介体桃蚜( Myzus persicae)的检测,则由于蚜虫体积小、RNA提取难度大、成本高、且不能复检,因而在生产中不能被广泛使用。该研究以马铃薯感病植株和带毒蚜虫为材料,利用改进的RNA提取方法从它们中提取到PLRV的RNA,并以CP 基因设计特异性引物,进行PCR检测。结果表明：该方法提取的RNA完整性好,可用于蚜虫中PLRV检测,且同样适用于对马铃薯感病植株的检测。另外,通过对田间有翅蚜和无翅蚜携带 PLRV 情况进行检测发现,无翅蚜 PLRV 检出率为100％,有翅蚜PLRV检出率也高达60％,证明该体系在生产中的实用性。该研究使用改进的RNA提取方法,提取蚜虫中RNA,并利用RT－PCR进行了PLRV检测,与以前的方法相比简单实用,可被应用于生产检测中。该研究结果为马铃薯生产中PLRV的防控提供了一种新的手段。     

Rapid and Efficient Gene Splicing Using Megaprimer-Based Protocol

Megaprimer-based methodology has been widely applied in site-directed mutagenesis, but rarely used in gene splicing. In this article, we describe a modification of the megaprimer PCR method, which can efficiently create and amplify a specific ligated chimeric gene segment in a PCR reaction and under a common PCR program that is widely used by researchers. More importantly, this modified method for splicing two or more gene fragments together revealed the mechanism of the megaprimer PCR method, by elucidating the key factor in the megaprimer-based protocol. In this method, the denatured megaprimer divided into two strands. One strand was used as template DNA to regenerate megaprimer and the other strand was used as an oligonucleotide primer to create a ligated chimeric gene product. In this article, we detail the modified megaprimer protocol for creating and amplifying these chimeric gene products, including a specific protocol for large chimeric gene products. We also provide additional tips to increase specificity and efficiency of the protocols. In conclusion, the improved megaprimer PCR protocol is a simple, broadly applicable protocol for splicing two different gene fragments together without relying on restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tunable Plasmonic Silver Nanodomes for Surface-Enhanced Raman Scattering

Surface-enhanced Raman scattering (SERS) is an emerging analytical method used in biological and non-biological structure characterization. Since the nanostructure plasmonic properties is a significant factor for SERS performance, nanostructure fabrication with tunable plasmonic properties are crucial in SERS studies. In this study, a novel method for fabrication of tunable plasmonic silver nanodomes (AgNDs) is presented. The convective-assembly method is preferred for the deposition of latex particles uniformly on a regular glass slide and used as a template for polydimethylsiloxane (PDMS) to prepare nanovoids on a PDMS surface. The obtained nanovoids on the PDMS are used as a mold for AgNDs fabrication. The nanovoids are filled with Ag deposition by the electrochemical method to obtain metallic AgNDs. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used for characterization of the structural properties of all fabricated AgNDs. The optical properties of AgNDs are characterized with the evaluation of SERS activity of 4-aminothiphonel and rhodamine 6G. In addition to experimental characterizations, the finite difference time domain (FDTD) method is used for the theoretical plasmonic properties calculation of the AgNDs. The experimental and theoretical results show that the SERS performance of AgNDs is strongly dependent on the heights and diameters of the AgNDs.     

A generalized estimating equations approach to quantitative trait locus detection of non-normal traits

To date, most statistical developments in QTL detection methodology have been directed at continuous traits with an underlying normal distribution. This paper presents a method for QTL analysis of non-normal traits using a generalized linear mixed model approach. Development of this method has been motivated by a backcross experiment involving two inbred lines of mice that was conducted in order to locate a QTL for litter size. A Poisson regression form is used to model litter size, with allowances made for under- as well as over-dispersion, as suggested by the experimental data. In addition to fixed parity effects, random animal effects have also been included in the model. However, the method is not fully parametric as the model is specified only in terms of means, variances and covariances, and not as a full probability model. Consequently, a generalized estimating equations (GEE) approach is used to fit the model. For statistical inferences, permutation tests and bootstrap procedures are used. This method is illustrated with simulated as well as experimental mouse data. Overall, the method is found to be quite reliable, and with modification, can be used for QTL detection for a range of other non-normally distributed traits.     

A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry

Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.     

A new microvolume technique for bioaffinity assays using two-photon excitation

Bioaffinity binding assays such as the immunoassay are widely used in life science research. In an immunoassay, specific antibodies are used to bind target molecules in the sample, and quantification of the binding reaction reveals the amount of the target molecules. Here we present a method to measure bioaffinity assays using the two-photon excitation of fluorescence. In this method, microparticles are used as solid phase in binding the target molecules. The degree of binding is then quantified from individual microparticles by use of two photon excitation of fluorescence. We demonstrated the effectiveness of the method using the human alpha-fetoprotein (AFP) immunoassay, which is used to detect fetal disorders. The sensitivity and dynamic range we obtained with this assay indicate that this method can provide a cost-effective and simple way to measure various biomolecules in solution for research and clinical applications.     

On-line Measurements and Control of Viable Cell Density in Cell Culture Manufacturing Processes using Radio-frequency Impedance

In this work, radio-frequency (RF) impedance is reviewed as a method for monitoring and controlling cell culture manufacturing processes. It is clear from the many publications cited that RF Impedance is regarded as an accurate and reliable method for measuring the live cell bio-volume both on-line and off-line and the technology is also sutable for animal cells in suspension, attached to micro-carriers or immobilized in fixed beds. In cGMP production, RF Impedance is being used in three main areas. Firstly, it is being used as a control instrument for maintaining consistent perfusion culture allowing the bioreactor to operate under optimum conditions for maximum production of recombinant proteins. In the second application it has not replaced traditional off-line live cell counting techniques but it is being used as an additional monitoring tool to check product conformance. Finally, RF Impedance is being used to monitor the concentration of live cells immobilized on micro-carriers or packed beds in cGMP processes where traditional off-line live cell counting methods are inaccurate or impossible to perform.     

A Simple and Reproducible Method for Directed Evolution: Combination of Random Mutation with dITP and DNA Fragmentation with Endonuclease V

An alternative method to combine mutagenesis PCR with dITP and fragmentation by endonuclease V for directed evolution was developed. In comparison to the routine protocol for directed evolution, dITP was used as mutation reagent in the mutagenesis PCR. Subsequently, the incorporated dITP in the PCR products could represent as being the target of endonuclease V. Finally, the mutated dsDNA was fragmented by endonuclease V and then shuffled via assembly and reamplification as is usually done. In this study, the gene encoding kanamycin resistance has been used as reporter to verify the novel method for directed evolution. However, the mutation frequency could be easily adjusted by the amount of dITP used in the mutagenesis PCR reaction. Besides, this protocol yielded the mutation types with an obvious bias to transition substitutions as the normal error-prone PCR did. Conclusively, this novel method for directed evolution has been demonstrated to be efficient, reproducible, and easy to handle in actual practice. Using this protocol, we have successfully constructed a random mutation library for the gene encoding a serine alkaline protease.     

Mass spectrometry analysis of blood low-molecular fraction as a method for unification of therapeutic drug monitoring

The review describes a new method of therapeutic drug monitoring (TDM) based on direct infusion of low-molecular fraction of blood into an electrospray ionization source of mass spectrometer. This technique allows performing TDM of almost all drugs used in clinical practice. Universality and a high-throughput mode of the method significantly simplify wide application of this method. Moreover, the possibility of method application in most cases of drug therapy has been argued as a tool for control of drug doses, rationality of drug therapy, and quality of drugs used. In conclusion, prospects for application of the method as primary means of improving the quality and personalization of drug therapy have been discussed.     

一种检测kras基因突变的新型TB-ARMS qPCR方法的建立

在荧光定量PCR基础上建立一种简单有效并且高度灵敏的TB-ARMSkras基因突变检测方法,并对其检测性能进行评估,探讨其临床应用价值。针对kras基因8种常见的点突变类型,通过设计并优化突变特异性引物、野生型特异性封闭引物并综合应用突变富集扩增反应条件等多种手段,提高点突变检测的灵敏度和特异性,采用已知野生型基因组样品和构建的突变质粒作为标准品,进行方法学评价;通过对临床样本的检测及与现有商品化试剂盒的比较进行性能验证;通过对术前血浆和配对组织样品的对比检测,评估方法是否适用于血液样本的检测。建立了TB-ARMS kras突变检测的新方法,能检测的最低突变率可达到0.01%。通过综合采用野生型特异性封闭引物和突变富集扩增条件等方法证明了其0.01%的突变检测灵敏度。检测准确性优于现有商品化试剂盒,血浆DNA TB-ARMS qPCR检测结果与配对组织DNA测序结果相符合。因此,TB-ARMS kras基因突变检测方法具有广泛的临床应用价值,既适用于临床组织样品的检测,也可应用于液体活检。     

A method for assessing the efficacy of using bifidumbacterin for the prevention of suppurative-inflammatory diseases in the newborn

A method has been developed for the evaluation of the effectiveness of bifidumbacterin in different quantitative morbidity characteristics in purulent inflammatory diseases of newborns in risk groups. This method requires a limited number of observations. In purulent inflammatory infections bifidumbacterin can be used as an effective remedy for the prophylaxis of hospital infections. The proposed method may be used for the analysis of the effectiveness of other antiepidemic measures, e.g. the sanitation of carriers.     

免疫-PCR法检测梅毒螺旋体特异性抗体

以梅毒螺旋体重组蛋白为抗原,应用免疫-PCR方法检测梅毒螺旋体抗体,并同常规ELISA法进行比较,探讨免疫-PCR方法检测梅毒螺旋体特异性抗体的可行性。结果免疫-PCR法敏感性是常规ELISA法的104倍,阳性检出率高于ELISA法;对照血清标本梅毒螺旋体抗体检测为阴性。表明免疫-PCR方法具有较高敏感性和特异性,有一定的临床推广价值,对梅毒患者的早期诊断及时治疗等具有重要意义。     

Improvement of the micromethod for the limulus lysate test.

Frauch's micro-slide method was improved to facilitate the endpoint-determination of the Limulus test. Two precise observations, by inverted phase contrast microscopy and with a staining procedure, were newly performed as additions to the slide test. The staining procedure was proposed as an improved method for the Limulus test since it is simple and convenient. In the staining method, bromophenol blue (BPB) solution was used as the staining solution. A negative (-), a strong positive (++) and a weak positive reaction (+) were characterized by a "ring" formation, a "cloud-like" spread of gel and a "spot" in the "cloud" respectively. Since the distinction between (-) and (+) reactions was obvious in the proposed method, determination of the endpoint was easier than in the ordinary tube and Frauch's method. The sensitivity of the present method was equal to or higher than that of other methods. Inverted phase contrast microscopy was utilized to confirm the findings obtained by the staining method. The volume of the lysate used in this method was as little as 1/10 of that used in the tube method.     

Gelatin-glutaraldehyde cross-linking on silicone rubber to increase endothelial cell adhesion and growth

Silicone is a biomaterial that is widely used in many areas because of its high optical clarity, its durability, and the ease with which it can be cast. However, these advantages are counterbalanced by strong hydrophobicity. Gelatin cross-linking has been used as a hydrophilic coating on many biomaterials but not on silicone rubber. In this study, two gelatin glutaraldehyde (GA) cross-linking methods were used to coat a hydrophilic membrane on silicone rubber. In method I, gelatin and GA were mixed in three different proportions (64:1, 128:1, and 256:1) before coating. In method II, a newly formed 5% gelatin membrane was cross-linked with a 2.5% GA solution. All coatings were hydrophilic, as determined from the measurement of contact angle for a drop of water on the surface. Bovine coronary arterial endothelial cells were shown to grow well on the surface modified by method II at 72 h. In method I, the cells grew well for gelatin-GA proportions of 64:1 and 128:1 at 72 h. No cell attachment on untreated silicone rubber was observed by the third d of seeding. The results indicated that both methods of gelatin-GA cross-linking provided a hydrophilic surface on silicone for endothelial cell adhesion and growth in vitro.     

Immunohistochemical detection of antibody in tissue sections of non-perfused and ex vivo-perfused organs using a tetrazolium alkaline phosphatase substrate

We have used nitroblue tetrazolium (NBT) as a color reagent to localize antibody-bound alkaline phosphatase in frozen tissue sections. In the method described, NBT is reduced to a stable black diformazan reaction product that contrasts well with nuclear counterstains such as hematoxylin and stands out strongly in black and white photographs. We have found NBT to be a suitable color reagent for the alkaline phosphatase: anti-alkaline immunohistochemical technique. The reaction product also contrasts well with fast red and can therefore be used as second reagent for two color immunoenzyme studies. In this report, we describe a novel two color immunoenzyme method to assess the ex vivo binding of antibodies against Class II histocompatibility antigens in whole organs connected to a perfusion circuit.     

Immunohistochemical detection of antibody in tissue sections of non-perfused and ex vivo-perfused organs using a tetrazolium alkaline phosphatase substrate

Summary We have used nitroblue tetrazolium (NBT) as a color reagent to localize antibody-bound alkaline phosphatase in frozen tissue sections. In the method described, NBT is reduced to a stable black diformazan reaction product that contrasts well with nuclear counterstains such as hematoxylin and stands out strongly in black and white photographs. We have found NBT to be a suitable color reagent for the alkaline phosphatase: anti-alkaline immunohistochemical technique. The reaction product also contrasts well with fast red and can therefore be used as second reagent for two color immunoenzyme studies. In this report, we describe a novel two color immunoenzyme method to assess the ex vivo binding of antibodies against Class II histocompatibility antigens in whole organs connected to a perfusion circuit.     

Hierarchical control of biotechnological complex systems

Application of hierarchical control for solution of some nonlinear optimization problems in fermentation systems, with the use of microprocessors, is described. Results presented show some advantages of the method of hierarchical control in comparison to some traditional one-level optimization methods. The advantage of the hierarchical control method is in less demand on memory and computing power of the control computer as the standard methods.In solving this problem two control schemes are used. In the first control scheme the method of objective coordination is used. In the second one the prediction method of coordination is used, with the aim to minimize selected disturbances in state and control variables.