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Showing papers in "BioTechniques in 2005"


Journal ArticleDOI
TL;DR: This review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for Relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries.
Abstract: Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.

1,739 citations


Journal ArticleDOI
TL;DR: A simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs and revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.
Abstract: MicroRNAs (miRNAs) are 20-24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. Several Arabidopsis miRNA sequences were tested using SYBR Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having asfew as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.

700 citations


Journal ArticleDOI
TL;DR: An overview of the common methods used in the fabrication of polymer microfluidic systems, including replica and injection molding, embossing, and laser ablation are presented.
Abstract: This review article describes recent developments in microfluidics, with special emphasis on disposable plastic devices. Included is an overview of the common methods used in the fabrication of pol...

503 citations


Journal ArticleDOI
TL;DR: Zymography is a valuable tool for research purposes and for the development of new diagnostic techniques and therapies for pathological conditions such as rheumatoid and osteoarthritis, and tumor progression.
Abstract: The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodeling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain degenerative diseases. To analyze the role of MMPs and TIMPs in tissue remodeling under normal and pathological conditions, it is important to have reliable detection methods. This review will focus on zymographical techniques for the analysis of MMPs and TIMPs. MMPs can be analyzed with several zymographical techniques, but substrate zymography is the most commonly used. This technique identifies MMPs by the degradation of their preferential substrate and by their molecular weight. Several substrates that can be used for zymography are described. Reverse zymography, which detects TIMPs by their ability to inhibit MMPs, is also discussed. Finally, in situ zymography is described, which is used to localize MMPs in tissue sections. Common problems encountered during sample preparation, zymography itself and the data analysis are discussed. Hints are given to improve the sensitivity and accuracy of zymographical methods. In conclusion, zymography is a valuable tool for research purposes and for the development of new diagnostic techniques and therapies for pathological conditions such as rheumatoid and osteoarthritis, and tumor progression.

429 citations



Journal ArticleDOI
TL;DR: The data indicate that the real-time PCR method is highly efficient with an efficiency coefficient close to the theoretical value of two and the second derivative method is more accurate than the fit point method in quantifying low gene expression levels.
Abstract: Quantification of mRNA expression levels using real-time reverse transcription PCR (RT-PCR) is increasingly used to validate results of DNA microarrays or GeneChips ® . It requires an improved method that is more robust and more suitable for high-throughput measurements. In this report, we compare a user non-influent, second derivative method with that of a user influent, fit point method that is widely used in the literature. We also describe the advantage of using a double correction: one correction using the expression levels of a housekeeping gene of an experiment as an internal standard and a second using reference expression levels of the same housekeeping gene in the tissue or cells. The first correction permits one to decrease errors due to sample preparation and handling, while the second correction permits one to avoid the variation of the results with the variability of housekeeping in each tissue, especially in experiments using various treatments. The data indicate that the real-time PCR method is highly efficient with an efficiency coefficient close to the theoretical value of two. The results also show that the second derivative method is more accurate than the fit point method in quantifying low gene expression levels. Using triplicate experiments, we show that measurement variations using our method are low with a mean of variation coefficients of <1%.

303 citations


Journal ArticleDOI
TL;DR: Validation of enumeration reveals correlation to be better than 0.98 when total bacterial counts by CellC are compared with manual enumeration, with all validated image types.
Abstract: Automated image analysis software, CellC, was developed and validated for quantification of bacterial cells from digital microscope images. CellC enables automated enumeration of bacterial cells, comparison of total count and specific count images [e.g., 4',6-diamino-2-phenylindole (DAPI) and fluorescence in situ hybridization (FISH) images], and provides quantitative estimates of cell morphology. The software includes an intuitive graphical user interface that enables easy usage as well as sequential analysis of multiple images without user intervention. Validation of enumeration reveals correlation to be better than 0.98 when total bacterial counts by CellC are compared with manual enumeration, with all validated image types. The software is freely available and modifiable: the executable files and MATLAB source codes can be obtained at www. cs. tut.fi/sgn/csb/cellc.

291 citations


Journal ArticleDOI
TL;DR: A condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection is described.
Abstract: The methylotrophic yeast Pichia pastoris has gained widespread acceptance as a system of choice for heterologous protein expression in part because of the simplicity of techniques required for its molecular genetic manipulation (1). Several different procedures are available for introducing DNA into P. pastoris—spheroplast generation (2), electroporation (3), alkali cation (3,4), or polyethylene glycol (PEG) treatment (5). Here we describe a condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection. The introduction of exogenous DNA into an organism requires two steps: (i) the preparation of competent cells for DNA uptake and (ii) the transformation of the cells with the DNA. Transformation of P. pastoris by electroporation is a quick procedure. However, preparation of conventional electroporation-competent cells requires hours of work involving several washes, incubations, and centrifugations. In contrast, competent cell preparation for the heat-shock method is short, but transformation requires approximately 2 h (4). The heat-shock procedure gives approximately 100-fold lower transformation efficiency than electroporation with plasmids containing auxotrophic marker genes such as HIS4. Additionally, the selection of zeocin-resistant transformants using the heat-shock transformation protocol does not work reliably. We have modified the preparation of competent cells from the heat-shock procedure (5) and combined it with transformation by electroporation (3) to yield a condensed protocol that works consistently with auxotrophic markers or antibiotic selection. The main modification of the heat-shock procedure is the addition of a step in which P. pastoris cells are incubated in an optimized concentration of dithiothreitol (DTT). The cells prepared by this “hybrid” method are then electroCondensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris

268 citations



Journal ArticleDOI
TL;DR: A novel ChIP assay is developed using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross- linking prior to conventional DNA-proteinCross-l linking, resulting in quantitative and robust identification of inducible NF-kappaB family binding to a variety of validated NF- kappaB-dependent genomic targets.
Abstract: The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this metho...

249 citations


Journal ArticleDOI
TL;DR: The two peer approaches to SDOCT are compared in speed, scan depth range, complexity, spectral regions of operation, and methods of detection and possible directions that this research may take in the near future are discussed.
Abstract: This paper reviews the current state of research in spectral domain optical coherence tomography (SDOCT). SDOCT is an interferometric technique that provides depth-resolved tissue structure information encoded in the magnitude and delay of the back-scattered light by spectral analysis of the interference fringe pattern. There are two approaches to SDOCT--one that uses a broadband source and a spectrometer to measure the interference pattern as a function of wavelength and the other that utilizes a narrowband tunable laser that is swept linearly in k approximately 1/lambda space during spectral fringe data acquisition. Unlike time domain (TD) OCT, the reference arm is stationary in both SDOCT methods, which allows for ultra high-speed OCT imaging. Owing to its high speed and superior sensitivity, SDOCT has become indispensable in biomedical imaging applications. After a brief introduction and a discussion on sensitivity advantage, methods of implementation of the two SDOCT schemes will be presented. The two peer approaches are compared in speed, scan depth range, complexity, spectral regions of operation, and methods of detection. The review also discusses OCT enhancements and functional methods based on SDOCT format and concludes with possible directions that this research may take in the near future.

Journal ArticleDOI
TL;DR: Substantial modifications and improvements are reported to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans.
Abstract: Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.

Journal ArticleDOI
TL;DR: Insight is provided into the current gene silencing techniques in mammalian systems and RNAi holds great promise as a novel therapeutic approach in cell-based and in vivo settings.
Abstract: Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for the functional annotation of the Cae-norhabditis elegans and Drosophila melanogaster genomes. Recent advances ...

Journal ArticleDOI
TL;DR: This review focuses on the current technology that takes advantage of the zebrafish model system to further the understanding of the genetic basis of cancer and its treatment.
Abstract: The zebrafish (Danio rerio) has proven to be a powerful vertebrate model system for the genetic analysis of developmental pathways and is only beginning to be exploited as a model for human disease and clinical research. The attributes that have led to the emergence of the zebrafish as a preeminent embryological model, including its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200–300 eggs per week, external fertilization that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, which allows the direct observation of developing internal organs and tissues in vivo. The zebrafish is amenable to transgenic and both forward and reverse genetic strategies that can be used to identify or generate zebrafish models of different types of cancer and may also present significant advantages for the discovery of tumor suppressor genes that promote tumorigenesis when mutationally inactivated. Importantly, the transparency and accessibility of the zebrafish embryo allows the unprecedented direct analysis of pathologic processes in vivo, including neoplastic cell transformation and tumorigenic progression. Ultimately, high-throughput modifier screens based on zebrafish cancer models can lead to the identification of chemicals or genes involved in the suppression or prevention of the malignant phenotype. The identification of small molecules or gene products through such screens will serve as ideal entry points for novel drug development for the treatment of cancer. This review focuses on the current technology that takes advantage of the zebrafish model system to further our understanding of the genetic basis of cancer and its treatment.

Journal ArticleDOI
TL;DR: It is shown that both single- and double-stranded DNA viruses can be recovered from blood samples using this approach and that the discovery of novel anellovirus sequences in the blood of healthy donors is reported.
Abstract: Rapid identification of viruses is needed to monitor the blood supply for emerging threats. Here we present a method that meets these criteria and allows for the shotgun sequencing of novel, uncultured DNA viruses directly from human blood. This method employs selection based on the physical properties of viruses combined with sequence-independent amplification and cloning. We show that both single- and double-stranded DNA viruses can be recovered from blood samples using this approach. In addition, we report the discovery of novel anellovirus sequences in the blood of healthy donors. PCR primers designed to amplify these novel anellovirus sequences were then used to verify the presence of these viruses in the general donor population.

Journal ArticleDOI
TL;DR: This simple conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 microm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating.
Abstract: Increasing numbers of cell mechanotransduction studies are currently utilizing elastic substrates fabricated from polyacrylamide in the form of thin gels. Their versatility depends on the ability to ensure the appropriate gel stiffness and control the uniformity and geometry of extracellular matrix protein coating of the gel. Beginning with a brief quantitative emphasis on the elastic properties of polyacrylamide gels, we present an inexpensive and highly reproducible method for uniform coating with a wide variety of extracellular matrix proteins. We used a reducing agent, hydrazine hydrate, to modify nonreactive amide groups in polyacrylamide to highly reactive hydrazide groups that can form covalent bonds with aldehyde or ketone groups in oxidized proteins. This simple conjugation method overcomes the limitations of previously used photoactivatable cross-linkers: nonuniform coating due to nonuniformity of irradiation and technically challenging procedures for micropatterning. As demonstrated in our study of cell polarity during constrained migration, this conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 μm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating.

Journal ArticleDOI
TL;DR: The use of HSV amplicon vectors to study Alzheimer disease, drug addiction, and depression are described, and the considerations that enter into the use of these vectors both in vitro and in vivo are discussed.
Abstract: There is an enormous initiative to establish the genetic basis for disorders of brain function. Unfortunately, genetic intervention is not accomplished easily in the nervous system. One strategy is...

Journal ArticleDOI
TL;DR: This renovated protocol saves time and effort and is cost-effective (by a dramatic reduction in the use of expensive SA-coated superparamagnetic beads), although additional materials are used.
Abstract: BENCHMARKS dsDNA was removed from the super-natant (lane 4), which was discarded. The bead-bound dsDNA was denatured in alkaline solution (0.1 M NaOH and 1 mM EDTA) at room temperature for 15 min, and the ssDNA without biotin groups was released in the alkaline solution (lane 5). After neutralization, the ssDNA solution was combined with five volumes of Buffer PB from the MiniElute™ PCR Purification Kit passed through MiniElute columns. The ssDNA was then eluted from the columns with a small volume of 10 mM Tris-HCl, pH 7.4 (lane 6). The recovery yield was 85% for this 149-nucleotide ssDNA. The whole procedure could be easily performed in 1 h either manually or by an automation workstation) with a robotic liquid handler, vacuum and manifold, washing unit, and a magnetic plate. Compared to the original protocols (1–3) or other standard methods involving gel purification and ethanol precipitation (4,5), this renovated protocol saves time and effort and is cost-effective (by a dramatic reduction in the use of expensive SA-coated superparamagnetic beads), although additional materials are used. The recovery yield of this protocol is a little lower than that (approximately 95%) of the original ones (1–3) because the binding affinity of silica membrane is weak for short DNA (approximately 75% for an 88-nucleotide ssDNA; data not shown) while alcohol precipitation can effectively recover short nucleic acids from solution. When short ssDNA (60–75 nucleotides) is to be prepared with high recovery yield, instead of using silica membrane, this protocol can be modified by using a molecular sieve column (Sephadex ® G-25; Amersham Biosci-ences, Piscataway, NJ, USA) to desalt. However, the volume of prior alkaline and acid solutions must be reduced to increase the ssDNA concentration.

Journal ArticleDOI
TL;DR: Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms.
Abstract: Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnost...

Journal ArticleDOI
TL;DR: The objective of this study was to analyze the influence of RNA degradation on the stability and expression pattern of different internal control genes in patients with nasal polyposis and chronic rhinosinusitis.
Abstract: Gene expression quantification methods are important tools in the understanding of the molecular events underlying human diseases and in the identification of diagnostic and therapeutic targets. Generally, the messenger RNA (mRNA) used for these analyses is derived from human biopsies obtained after surgery. As a consequence, several steps during tissue handling have to be carefully controlled in order to preserve the quality and integrity of the RNA material. It is well known that RNA is sensitive to degradation by postmortem processes and inadequate sample handling or storage (1). However, RNA integrity control is often not systematically performed prior to (PCR-based) downstream analyses. While in the past, RNA quality could often not be assessed due to the limited availability of the precious sample (e.g., from microdisected cells or small biopsies), the advent of capillary gel electrophoresis and (sample retention) spectrophotometry technologies (e.g., NanoDrop® ND-1000; NanoDrop Technologies, Wilmington, DE, USA ) has addressed this issue, allowing quality estimations using only nanograms (or even picograms) of total RNA (2). In addition, amplification of RNA is now an alternative method to obtain sufficient amounts to conduct gene expression studies when postmortem tissues are scarce; however, assessment of RNA quality based on the 18S and 28S ribosomal RNA bands is often not possible anymore after amplification. Furthermore, it remains to be determined whether the amplified mRNA can faithfully be used to assess RNA quality of the starting material. Apart from RNA quality, the choice of a proper set of reference genes for accurate normalization is another crucial factor with a profound impact on the reliability of the obtained gene expression levels (3). Reference genes are expressed constitutively in every cell; however, their expression can be regulated with diseases state, during cellular proliferation, due to cellular composition and by mitogenic stimuli (e.g., growth factors) (4,5). Furthermore, it is now known that life styles and genetic make-up of individuals can influence mRNA expression (6). That is why the validation of the expression stability of reference genes remains an important step to ensure the accuracy and reliability of gene expression studies. The objective of this study was to analyze the influence of RNA degradation on the stability and expression pattern of different internal control genes. To this purpose, 10 commonly used reference genes were quantified in both intact and degraded RNA from clinical specimens obtained from ethmoidal and maxillary sinuses collected from patients with nasal polyposis (NP) and chronic rhinosinusitis (CRS). Sixteen clinical tissue samples (30 mg) were homogenized in Tri-reagent buffer (Sigma, St. Louis, MO, USA) (1 mL/50–100 mg of tissue) in a chilled pestle mortar. Total RNA isolation and cDNA synthesis were performed as described previously (7). RNA Impact of RNA quality on reference gene expression stability

Journal ArticleDOI
TL;DR: This article introduces the new ClinProTools bioinformatics software, which performs all major steps of profiling, screening, and monitoring applications in clinical proteomics, and focuses on ClinProTool's powerful and intuitive visualization options forclinical proteomics applications.
Abstract: Recently, applications of mass spectrometry in the field of clinical proteomics have gained tremendous visibility in the scientific and clinical community One major objective is the search for pot


Journal ArticleDOI
TL;DR: An improved method for preparing cell aggregates for in vitro chondrogenesis studies and a modification of a previously developed conical tube-based culture system that replaces the original 15-mL polypropylene tubes with 96-well plates demonstrate morphological characteristics that are consistent with chONDrogenic differentiation and cartilaginous extracellular matrix production.
Abstract: We have developed an improved method for preparing cell aggregates for in vitro chondrogenesis studies. This method is a modification of a previously developed conical tube-based culture system that replaces the original 15-mL polypropylene tubes with 96-well plates. These modifications allow a high-throughput approach to chondrogenic cultures, which reduces both the cost and time to produce chondrogenic aggregates, with no detrimental effects on the histological and histochemical qualities of the aggregates. We prepared aggregates in both systems with human bone marrow-derived mesenchymal stem cells (hMSC). The aggregates were harvested after 2 and 3 weeks in chondrogenic culture and analyzed for their ability to differentiate along the chondrogenic pathway in a defined in vitro environment. Chondrogenic differentiation was assessed biochemically by DNA and glycosaminoglycan (GAG) quantification assays and by histological and immunohistologic assessment. The chondrogenic cultures produced in the 96-well plates appear to be slightly larger in size and contain more DNA and GAG than the aggregates made in tubes. When analyzed histologically, both systems demonstrate morphological characteristics that are consistent with chondrogenic differentiation and cartilaginous extracellular matrix production.

Journal ArticleDOI
TL;DR: A novel statistical method, based on linear mixed-effects models, is developed that finds that real-time RT-PCR using TaqMan Low-Density Arrays yielded reproducible measurements over seven orders of magnitude.
Abstract: Profiling studies using microarrays to measure messenger RNA (mRNA) expression frequently identify long lists of differentially expressed genes. Differential expression is often validated using rea...

Journal ArticleDOI
TL;DR: This study combines the sacB-based negative selection system with a cre-lox antibiotic marker recycling method, which can create P. aeruginosa strains with multiple mutations without modifying the antibiotic resistance profile when compared to the parental strain.
Abstract: The complete genome of the bacterial pathogen Pseudomonas aeruginosa has now been sequenced, allowing gene deletion, one of the most frequently used methods in gene function study, to be fully exploited. In this study, we combine the sacB-based negative selection system with a cre-lox antibiotic marker recycling method. This methodology allows allelic exchange between a target gene and a gentamicin cassette flanked by the two lox sequences. A tetracycline plasmid expressing the cre recombinase is then introduced in the mutant strain to catalyze the excision of the lox-flanked resistance marker. We demonstrate here the efficiency of the combination of these two methods in P. aeruginosa by successively deleting ExoS and ExoT, which are two genetically independent toxins of the type-three secretion system (TTSS). This functional cre-lox recycling antibiotic marker system can create P. aeruginosa strains with multiple mutations without modifying the antibiotic resistance profile when compared to the parental strain.

Journal ArticleDOI
TL;DR: Invention in biomolecule ionization and matrix-assisted laser desorption/ionization placed mass spectrometry (MS) at the forefront of this emerging discipline and these techniques are generally credited for expanding the field substantially.
Abstract: Proteins are involved in nearly every aspect of cellular function. In fact, the characterization of proteins has become such a significant part of modern biology, it has inspired a new discipline: proteomics—the classification of the protein complement expressed by the genome of an organism. Technology development has driven, and continues to drive, rapid evolution in this field. Seventeen years ago innovations in biomolecule ionization [electrospray (1) and matrix-assisted laser desorption/ionization (2), ESI and MALDI, respectively] placed mass spectrometry (MS) at the forefront of this emerging discipline. Although biological MS was pursued prior to this work, these techniques are generally credited for expanding the field substantially. With these tools, mass spectrometrists could for the first time, easily and robustly, convert condensed phase peptides or even whole proteins to intact gas phase ions.

Journal ArticleDOI
TL;DR: It is hoped that newly discovered cancer biomarkers and advances in high-throughput technologies would revolutionize cancer therapies by improving cancer risk assessment, early detection, diagnosis, prognosis, and monitoring therapeutic response.
Abstract: In spite of advances in diagnostics and therapeutics, cancer remains the second leading cause of death in the U.S. Successful cancer treatment depends not only on better therapies but also on improved methods to assess an individual's risk of developing cancer and to detect cancers at early stages when they can be more effectively treated. Current cancer diagnostic imaging methods are labor-intensive and expensive, especially for screening large asymptomatic populations. Effective screening strategies depend on methods that are noninvasive and detect cancers in their early stages of development. There is increasing interest and enthusiasm in molecular markers as tools for cancer detection and prognosis. It is hoped that newly discovered cancer biomarkers and advances in high-throughput technologies would revolutionize cancer therapies by improving cancer risk assessment, early detection, diagnosis, prognosis, and monitoring therapeutic response. These biomarkers will be used either as stand-alone tests or to complement existing imaging methods.

Journal ArticleDOI
TL;DR: The results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples, and should also have broad application for various other types of environmental samples.
Abstract: The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.

Journal ArticleDOI
TL;DR: One enzymatic approach in particular using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust and will allow mutation detection to become an integral component of individualized medicine.
Abstract: Mutation is as necessary for life as fidelity is in DNA replication. The study of mutations reveals the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Indeed, recent mutations that have not yet become polymorphisms are often deleterious and pertinent to the disease history of afflicted individuals. This review discusses the principles behind a variety of methods for the detection of mutations and factors that should be considered in future methods design. One enzymatic approach in particular, using orthologs of the CEL I nuclease that show high specificity for all mismatches, appears to be easy and robust. Further developments of this and other methods will allow mutation detection to become an integral component of individualized medicine.

Journal ArticleDOI
TL;DR: This study presents a strategy for the optimized design of protein subfragments for subsequent antibody production, selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity.
Abstract: Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.