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Showing papers in "BioTechniques in 2000"


Journal ArticleDOI
TL;DR: The HotSHOT method is rapid, inexpensive and may be carried out in 96-well plates, making it amenable to automation and high-throughput genotyping, and finds it remarkably consistent.
Abstract: Preparation of mouse genomic DNA for PCR is costly and laborious, primarily because of the difficulty in physically separating DNA from other tissue components. This level of purification is unnecessary for many PCR applications. A simple alternative, lysis in an alkaline reagent and neutralization with a suitable buffer, has been used to prepare genomic DNA from buccal swabs, whole blood, semen and forensic samples collected from humans (2). A few modifications of this protocol make it possible to prepare PCR-quality mouse genomic DNA with a brief incubation in hot sodium hydroxide and pH adjustment with a Tris solution (HotSHOT). The HotSHOT method is rapid, inexpensive and may be carried out in 96-well plates, making it amenable to automation and high-throughput genotyping. The reagents for HotSHOT DNA preparation are simple to prepare. An alkaline lysis reagent with 25 mM NaOH, 0.2 mM disodium EDTA and a pH of 12 is prepared by dissolving the salts in water without adjusting the pH. A neutralizing reagent with 40 mM Tris-HCl and a pH of 5 is prepared by dissolving Tris-HCl (not Tris base) in water without adjusting the pH.Tissue samples (neonatal mouse toes, ear punches, 0.2-cm tail snips or 25-mg pieces of spleen) are collected into a 96-well thermal cycler plate or thermal cycler strip tubes. The tissue sample must be small; too large a sample can cause the method to fail. Alkaline lysis reagent (75 μL is added to the samples and heated to 95°C for 10 min to 1 h. The undissolved tissue does not interfere with PCR. After heating, samples are cooled to 4°C, and 75 μL neutralizing reagent are added to each sample. One to five microliters of the final preparation are used per each 10-μL PCR volume. The combination of the alkaline lysis and neutralizing reagents yields a buffer consisting of 20 mM Tris-HCl (pH 8.1) and 0.1 mM EDTA, which is similar to a common DNA storage buffer. We have used this technique to prepare DNA for hundreds of PCR assays and find it remarkably consistent. Based on our initial success, we tested the method with tissues commonly used as a source of mouse genomic DNA. Toes from neonatal mice, 0.2-cm tail snips from weanling mice, ear punches from adult mice and small pieces of spleen (about 25 mg) from adult mice were placed in thermal cycler strip tubes with 75 μL alkaline lysis reagent added to each sample. Samples were heated to 95°C for 10, 20, 30 or 60 min, chilled to 4°C and 75 μL neutralizing reagent were added to each tube. The yield of soluble DNA was measured by a fluorometric assay (1). The performance of HotSHOT DNA in PCR was tested by amplifying a 182bp PCR product and comparing the performance of DNA prepared by proteinase K digestion, followed by phenol:chloroform extraction and ethanol precipitation (3). In this experiment, 1.5 μL HotSHOT DNA was combined with 8.5 μL PCR mixture. Forward and reverse primers were combined at 200 nM each with PCR buffer, 3 mM MgCl2, 0.2 mM each dATP, dCTP, dGTP and dTTP, 0.12 U Taq DNA polymerase (Promega, Madison, WI, USA), in a 10-μL volume. Reactions were covered with 10 μL mineral oil and amplified in a PTC-100 thermal cycler (MJ Research, Watertown, MA, USA) for 40 cycles at 95°C for 40 s and 60°C for 45 s. Following amplification, 10 μL loading dye were added to each well (4 μL 60% sucrose and 1 mM cresol red plus 6 μL water). Twelve microliters of the reaction were loaded on a 12% vertical polyacrylamide mini-gel (8 × 10 cm) and electrophoresed at 165 V for 45 min in 1× TBE buffer (89 mM Tris/89 mM boric acid/2 mM EDTA). Gels were stained for about 2 min in 1× TBE containing 1 μg/mL ethidium bromide and imaged under UV light using an Ambis Imaging System (Scanalytics, Billerica, MA, USA). The yield of soluble DNA varied with the tissue type and heating time (Figure 1). The yield was greatest from spleen. Ear punches, neonatal toes and tail snips produced similar amounts of soluble DNA. The yield of DNA increased with heating time for each of these tissues, most dramatically with spleen. However, the variation in soluble DNA among tissues and heating times had little influence on the amplification of the PCR product (Figure 2). These results suggest that the duration of heating time is not critical to the reaction outBenchmarks

1,413 citations


Journal ArticleDOI
TL;DR: The Sequence Manipulation Suite is a collection of freely available JavaScript applications for molecular biologists that consists of over 30 utilities for analyzing and manipulating sequence data, including the following.
Abstract: JavaScript is an object-based scripting language that can be interpreted by most commonly used Web browsers, including Netscape® Navigator® and Internet Explorer®. In conjunction with HTML form elements, JavaScript can be used to make flexible and easy-to-use applications that can be accessed by anyone connected to the Internet (3). The Sequence Manipulation Suite (http://www.ualberta.ca/~stothard/javascript/) is a collection of freely available JavaScript applications for molecular biologists. It consists of over 30 utilities for analyzing and manipulating sequence data, including the following:

1,299 citations


Journal ArticleDOI
TL;DR: This work has optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, probe labeling and hybridization, and has developed strategies for data normalization and analysis.
Abstract: Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, probe labeling and hybridization, and have developed strategies for data normalization and analysis.

1,017 citations


Journal ArticleDOI
TL;DR: The uses and pitfalls of the most popular of these controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, are discussed, with special emphasis on precautions associated with the use of GAPDH.
Abstract: The study of mammalian gene expression is often carried out at the level of mRNA. In such analyses, one usually measures the amount of an mRNA of interest under different conditions such as stress,...

869 citations


Journal ArticleDOI
TL;DR: This method of temporal transfection might have utility in reversing cellular abnormalities through genetic intervention of the temporally introduced gene.
Abstract: the fact that ECs are difficult to transfect, the success of this method is warranted for a wider range of cells with different origins. In fact, we have obtained similar results in temporally transfecting NIH 3T3 cells using this method (data not shown). In addition, this method of temporal transfection might have utility in reversing cellular abnormalities through genetic intervention of the temporally introduced gene.

351 citations



Journal ArticleDOI
TL;DR: A significantly improved method allows the use of a single primer to generate deletions exceeding 3000 bp and point mutations in a single linear amplification reaction.
Abstract: Site-directed mutagenesis is an important technique for studying DNA and protein function. With the advent of PCR, several new approaches have been successfully developed (1,3,5–9). Early methods involved two-step PCR followed by subcloning, which is time consuming and error prone. Recently introduced site-directed mutagenesis kits such as QuikChangeTM Site-Directed Mutagenesis Kit (QCS) developed by Stratagene (La Jolla, CA, USA) is a widely used PCR-based system (4) that eliminates the necessity to subclone the amplified DNA fragment. It requires two complementary mutagenic oligonucleotide primers and uses a plasmid to amplify the desired product by highfidelity Pfu DNA polymerase (Stratagene). Following temperature cycling, the product is treated by the restriction endonuclease DpnI (Stratagene) that will cut only fully or hemimethylated 5′-Gm6ATG-3′ DNA sequences. Thus, only the original parental methylated plasmid DNA will be digested. The remaining unmethylated and nicked double-stranded plasmid incorporating the desired mutations is then transformed into Epicurian Coli® XL-1Blue supercompetent cells (Stratagene). The yield of clones harboring mutations is very high and varies, depending on the template length, from 80% to more than 90% for 2.9-kb plasmids (4). Methods for generating internal deletions have been described by using inverse PCR, but these involved two steps (9). A simplified method for deletion mutagenesis is used in the ExSiteTM mutagenesis kit by Stratagene, but this method also involves the use of phosphorylated primers and a ligation step. The use of the QuikChange mutagenesis kit has been reported for introducing deletions or insertions of only 12 bp (4). Here, we present a significantly improved method based on the QuikChange system for generating deletion and point mutants. This method allows the use of a single primer to generate deletions exceeding 3000 bp and point mutations in a single linear amplification reaction. In our laboratory, we routinely use the QuikChange kit for point mutations. However, it was not clear how this protocol could be applied for deletions of more than 12 bp (4). We used a plasmid containing Pals1 cDNA (total length 6.7 kb) as a template to make several deletion and point mutants (2). The PCR mutagenesis reaction was performed in a reaction volume of 25 μL that included 1 μL plasmid DNA (in TE or water) from Wizard® Plus SV Minipreps DNA Purification System by Promega (Madison, WI, USA) or from QIAprep® Spin miniprep kit by Qiagen (Valencia, CA, USA) miniprep (100– 300 ng/μL) template DNA, 200 nM each primer, 200 μM dNTPs and 1.25 U Pfu enzyme in 1× Pfu DNA polymerase reaction buffer. A preliminary step of denaturation at 95°C for 3 min was followed by 18 cycles of PCR. These PCR cycles consisted of 15 s of denaturation at 95°C, 1 min of annealing at a temperature 2°C higher than the melting temperature (Tm) of the head of the primer to increase specificity of amplification (see below) and 12 min of extension at 68°C using a PTC200TM thermal cycler (MJ Research, Watertown, MA, USA). In cases when the Tm exceeded the extension temperature, we applied a two-step PCR annealing and extending of 68°C. Finally, the PCR product was treated with 1 μL DpnI endonuclease for 2 h at 37°C and 2 μL DpnI-digested DNA was transformed into 50 μL Epicurean Coli XL1 Blue supercompetent cells. The number of kanamycin-resistant clones varied from 100 to 2000. We experimented with primer pairs that would result in the highest yield of the desired deletion. We routinely analyzed clones by PCR using primers flanking the deletions and obtained efficiencies of between 25% and 70%. Selected clones were sequenced and confirmed the PCR results. Table 1 summarizes different examples of primer pairs or single primers used in the mutagenesis experiments. For simplicity, the 5′ part of the primers complementary to the region upstream of the introduced deletion we called the tail, and the 3′ portion of the primer complementary to the region downstream of the deletion we called the head. As shown in Table 1, when using primer pairs of different design, deletions of up to 3 kb could be introduced into the plasmid by this simple one-step protocol. We believe that the same approach could be applied to any sequence because we successfully deleted several regions of the coding Benchmarks

225 citations


Journal ArticleDOI
TL;DR: The results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages.
Abstract: Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.

210 citations


Journal ArticleDOI
TL;DR: The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to the understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies.
Abstract: The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

199 citations


Journal ArticleDOI
TL;DR: This review will cover the status of the hybridization array field with an eye toward underlying principles and available technologies as well as future developments and technological trends.
Abstract: DNA hybridization arrays [also known as macroarrays, microarrays and/or high-density oligonucleotide arrays (Gene Chips)] bring gene expression analysis to a genomic scale by permitting investigators to simultaneously examine changes in the expression of literally thousands of genes. For hybridization arrays, the general approach is to immobilize gene-specific sequences (probes) on a solid state matrix (nylon membranes, glass microscope slides, silicon/ceramic chips). These sequences are then queried with labeled copies of nucleic acids from biological samples (targets). The underlying theory is that the greater the expression of a gene, the greater the amount of labeled target, and hence, the greater output signal. In spite of the simplicity of the experimental design, there are at least four different platforms and several different approaches to processing and labeling the biological samples. Moreover, investigators must also determine whether they will utilize commercially available arrays or generate their own. This review will cover the status of the hybridization array field with an eye toward underlying principles and available technologies. Future developments and technological trends will also be evaluated.

188 citations


Journal ArticleDOI
TL;DR: This review specifically focuses on what is now called antibody engineering and includes chimeric and humanized antibodies, immunoglobulin fragments, antibody libraries, antibody fusion proteins and transgenic organisms as bioreactors.
Abstract: It has been almost 100 years since von Behring and Kitasato received the first Nobel prize for the discovery of passive immunotherapy and nearly 25 years since Kohler and Milstein first reported hybridoma technology. In the 15 years since Mullis and co-workers described PCR, a number of discoveries and technologies have converged to produce a renaissance in antibody therapeutics. Our vision of antibodies as tools for research--useful for the prevention, detection and treatment of disease--has been revolutionized by these recent advances. This review specifically focuses on what is now called antibody engineering and includes chimeric and humanized antibodies, immunoglobulin fragments, antibody libraries, antibody fusion proteins and transgenic organisms as bioreactors. As a consequence of refinements in antibody technology, the field of genetically engineered immunoglobulins has matured into an elegant and important drug and reagent development platform.

Journal ArticleDOI
TL;DR: This work describes a system obtained by adapting a commercial ink-jet printer and used to produce mono- and bidimensional arrays of spots containing horseradish peroxidase on cellulose paper, which revealed that the reproducibility of the enzyme deposition was satisfactory for analytical purposes.
Abstract: Many recent bioanalytical systems based on immunologic and hybridization reactions in a mono- or bidimensional microarray format require technology that can produce arrays of spots containing biospecific molecules. Some microarray deposition instruments are commercially available, and other devices have been described in recent papers. We describe a system obtained by adapting a commercial ink-jet printer and used to produce mono- and bidimensional arrays of spots containing horseradish peroxidase on cellulose paper. In a few minutes, it was possible to obtain bidimensional arrays containing several thousands of spots with a diameter as low as 0.2 mm, with each of which requiring only a few nanoliters of the enzyme deposition solution. The quantity of enzyme in each spot was evaluated with a chemiluminescent reaction and a charge-coupled device-based, low-light imaging luminograph. The chemiluminescence measurements revealed that the reproducibility of the enzyme deposition was satisfactory for analytical purposes, with the variation coefficients being lower than 10% in almost all cases.

Journal ArticleDOI
TL;DR: Comparisons of the protein expression patterns from microdissected normal and tumor tissues indicated several differences, highlighting the importance of extremely defined tissue lysates for protein profiling.
Abstract: Analysis of whole genomes to monitor specific changes in gene activation or changes in gene copy number due to perturbation has recently become possible using DNA chip technologies. It is now becoming apparent, however, that knowing the genetic sequence encoding a protein is not sufficient to predict the size or biological nature of a protein. This can be particularly important in cancer research where posttranslational modifications of a protein can specifically lead to the disease. To address this area, several proteomic tools have been developed. Currently the most widely used proteomics tool is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which can display protein expression patterns to a high degree of resolution. However, 2D-PAGE can be time consuming; the analysis is complicated and, compared with DNA techniques, is not very sensitive. Although some of these problems can be alleviated by using high-quality homogeneous samples, such as those generated using microdissection technique...

Journal ArticleDOI
TL;DR: A one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses and its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enterovirus RNA.
Abstract: We have developed a quantitative RTPCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man® technology with the ABI PrismTM 7700...

Journal ArticleDOI
TL;DR: A synthetic fragment was added to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase that displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme.
Abstract: Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated, the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.

Journal ArticleDOI
TL;DR: It is demonstrated that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.
Abstract: Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.


Journal ArticleDOI
TL;DR: The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.
Abstract: PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.

Journal ArticleDOI
TL;DR: FAST slides have a much higher binding capacity for DNA and better spot-to-spot consistency than traditional poly-lysine-coated slides and are well suited for fluorescent detection because of their relatively low light scatter and efficient retention of arrayed DNA.
Abstract: We have evaluated FASTslides, a glass slide with a microporous polymeric surface that is a suitable substrate for microarray technology. The surface is a nitrocellulose-based polymer that binds DNA...

Journal ArticleDOI
I. Täpp1, L. Malmberg1, E. Rennel1, M. Wik1, Ann-Christine Syvänen1 
TL;DR: Two homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs) using three SNPs in the human estrogen receptor gene as targets.
Abstract: Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance ...

Journal ArticleDOI
TL;DR: The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to quantitative analysis of SNPs demonstrates that allele frequencies as low as 5% can be detected quantitatively and unambiguously.
Abstract: Pooling of DNA samples before genotyping is a valuable means of streamlining large-scale genotyping efforts in disease association studies, single-nucleotide polymorphism (SNP) validation or mutant allele screening programs. In this report, we explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantitative analysis of SNPs. The measurements are based on MALDI-TOF MS analysis of primer extension assays performed on standard mixtures of pooled PCR products at several test loci. The inherent high molecular weight resolution of MALDI-TOF MS conveys high specificity and good signal-to-noise ratio for performing accurate quantitation. The methods described maximize the sensitivity and quantitative capacity of MALDI-TOF MS while preserving the throughput and economic advantages of the MALDI-TOF platform. Using the format described, we demonstrate that allele frequencies as low as 5% can be detected quantitatively and unambiguously.

Journal ArticleDOI
TL;DR: To monitor transcript abundance in cDNA, both from nuclear run-on and total RNA, the proposed semiquantitative PCR approach based on the use of fluorescent primers is proposed.
Abstract: In this report, we present a fluorescence-based approach to the assessment of cellular gene expression and transcription rates. Nuclear run-on was performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads. Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. To monitor transcript abundance in cDNA, both from nuclear run-on and total RNA, we propose a semiquantitative PCR approach based on the use of fluorescent primers.

Journal ArticleDOI
TL;DR: The experiments presented here suggest that catabolite repression can effectively reduce basal expression of the T7 polymerase gene in BL21 (DE3), yielding tight regulation and consistency comparable to that of BL21(DE3) pLysS.
Abstract: The T7 polymerase-based pET System is one of the most powerful and widely used prokaryotic expression systems available today Expression of even slightly toxic gene products in BL21 (DE3), however, has been problematic due to basal expression, which leads to decreased plasmid stability and variable yields following large-scale growth and induction Use of host strains such as BL21 (DE3) pLysS provides high stringency and consistent expression but typically at the cost of reduced protein levels upon induction The experiments presented here suggest that catabolite repression can effectively reduce basal expression of the T7 polymerase gene in BL21 (DE3), yielding tight regulation and consistency comparable to that of BL21 (DE3) pLysS By switching to a poor carbon source for the final growth cycles, the higher expression levels typical of BL21 (DE3) can readily be obtained upon induction

PatentDOI
TL;DR: In this article, the use of aptamers in high throughput screening methods to determine whether a non-nucleic acid molecule, i.e. small molecule, binds to a target was discussed.
Abstract: The invention relates to the use of aptamers in high throughput screening methods to determine whether a non-nucleic acid molecule, i.e. small molecule, binds to a target.

Journal ArticleDOI
TL;DR: A technique that does not require cloning and allows rapid identification of transposon insertion sites using a single PCR primer and a uniquely designed PCR procedure is reported.
Abstract: Transposons have been used in many laboratories worldwide as a powerful tool for insertional mutagenesis to investigate gene function in bacteria (8). The technique has recently been adapted in which a transposon, such as mini-Tn5, delivers a unique identifying sequence of 40 nucleotides (1). This technique, termed signature-tagged mutagenesis (STM), is widely used in the search for virulence-associated genes via negative selection and allows the large-scale analysis of pooled mutants. Different procedures have been developed for identification of the transposon insertion regions. Single-primer PCR (4) requires restriction endonuclease digestion, ligation, PCR and cloning steps. It also depends on the availability of appropriate restriction sites. The rapid amplification of transposon ends (RATE) technique (6) involves several steps including ligation of DNA fragments to linkers, singlestrand amplification using biotin-labeled primers, isolation of the amplified products and another PCR before the sequencing. A single-primer PCR technique was originally developed by Parks et al. (5) and modified by Spector et al. (7). However, both techniques required intermediate cloning and selection steps before sequencing. Here, we report a technique that does not require cloning and allows rapid identification of transposon insertion sites using a single PCR primer and a uniquely designed PCR procedure. To test this method, we used mini-Tn5 insertion mutants from a recently constructed Yersinia pseudotuberculosis STM library (3). Figure 1 outlines the strategy for sequencing the transposon insertion regions. The procedure involves just one transposon-specific primer and a single PCR consisting of three rounds of amplification. The first round is designed for linear amplification of single-stranded, transposon-specific templates from the transposon that are to be used in the following steps and is carried out at a standard annealing temperature (50°C). The second round involves a low annealing temperature (30°C) for amplification of the region adjacent to the transposon insertion site. Amplification of this region is possible because, at low annealing temperatures, the primer will bind to sites of limited sequence comBenchmarks

Journal ArticleDOI
TL;DR: Both radiation-based methods for the determination of SNP allele frequencies gave accurate and reproducible results, suggesting they are suitable for use in DNA pooling experiments.
Abstract: Single nucleotide polymorphisms (SNPs) are among the most common types of polymorphism used for genetic association studies. A method to allow the accurate quantitation of their allele frequencies from DNA pools would both increase throughput and decrease costs for large-scale genotyping. However, to date, most DNA pooling studies have concentrated on the use of microsatellite polymorphisms. In the case of SNPs that are restriction fragment length polymorphisms (RFLPs), studies have tended to use methods for the quantitation of allele frequency from pools that rely on densitometric evaluation of bands on an autoradiograph. Radiation-based methods have well-known drawbacks, and we present two alternative methods for the determination of SNP allele frequencies. For RFLPs, we used agarose gel electrophoresis of digested PCR products with ethidium bromide staining combined with densitometric analysis of gel images on a PC. For all types of SNP, we used allele-specific fluorescent probes in the Taqman assay to determine the relative frequencies of two different alleles. Both methods gave accurate and reproducible results, suggesting they are suitable for use in DNA pooling experiments.


Journal ArticleDOI
TL;DR: The subject matter will address details of the methods used to produce images of cells and tissues at any magnification and resolution, and might include “tricks-of-the-trade”, novel methods of specimen preparation, practices of image collection, tips on the digital manipulation and publication of images and historical perspectives.
Abstract: feature short articles devoted to microscopy and digital imaging. The subject matter will address details of the methods used to produce images of cells and tissues at any magnification and resolution, and might include “tricks-of-the-trade”, novel methods of specimen preparation, practices of image collection, tips on the digital manipulation and publication of images and historical perspectives.

Journal ArticleDOI
TL;DR: In this paper, the feasibility of microcapsules based on hydrogels for transplantation of non-autologous cells and tissue fragments was shown, and the first successful long-term clinical applications were reported.
Abstract: Many diseases are closely tied to deficient or subnormal metabolic and secretory cell functions. Milder forms of these diseases can be managed by a variety of treatments. However, it is often extremely difficult or even impossible to imitate the moment-to-moment fine regulation and the complex roles of the hormone, factor or enzyme that is not sufficiently produced by the body. Immunoisolated transplantation is one of the most promising approaches to overcome the limitations of current treatments. Non-autologous (transformed) cell lines and allogeneic and xenogeneic cells/tissues that release the therapeutic substances are enclosed in immunoprotective microcapsules. The microcapsules avoid a lifetime of immunosuppressive therapy while excluding an immune response in the host. Research in this direction has shown the feasibility of microcapsules based on hydrogels (particularly of alginate) for transplantation of non-autologous cells and tissue fragments. Numerous technical accomplishments of the immunoisolation method have recently made possible the first successful long-term clinical applications. However, realizing the potential of immunoisolated therapy requires the use of several factors that have received limited attention in the past but are important for the formulation of hydrogel-based immunoisolation systems that are highly versatile, potentially economical and can gain medical approval.

Journal ArticleDOI
TL;DR: This work reports on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells and describes the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker.
Abstract: Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested vi...