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Molecular Biology Techniques (molecular + biology_techniques)
Selected AbstractsA research project-based and self-determined teaching system of molecular biology techniques for undergraduates,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 3 2008Shuping Zhang Abstract Molecular biology techniques play a very important role in understanding the biological activity. Students who major in biology should know not only how to perform experiments, but also the reasons for performing them. Having the concept of conducting research by integrating various techniques is especially important. This paper introduces a research project-based and self-determined teaching system of molecular biology techniques for undergraduates. Its aim is to create an environment mimicking real research programs and to help students build up confidence in their research skills. The students are allowed to explore a set of commonly used molecular biology techniques to solve some fundamental problems about genes on their own. They find a gene of interest, write a mini-proposal, and give an oral presentation. This course provides students a foundation before entering the research laboratory and allows them to adapt easily to real research programs. [source] THE MOLECULAR FUTURE IN CYTOLOGYCYTOPATHOLOGY, Issue 2006M. Salto-Tellez Molecular diagnosis is the application of molecular biology techniques and knowledge of the molecular mechanisms of disease to diagnosis, prognostication and treatment of diseases. Molecular Diagnosis is, arguably, the fastest growing area of diagnostic medicine. The US market for molecular testing generated $1.3 billion in 2000, which was predicted to increase to about $4.2 billion by 2007.1 We proposed the term Diagnostic Molecular Cytopathology to define the application of molecular diagnosis to cytopathology2. Diagnostic Molecular Cytopathology is essential for the following reasons: (i) Molecular testing is sometimes indispensable to establish an unequivocal diagnosis on cell preparations; (ii) Molecular testing provides extra information on the prognosis or therapy of diseases diagnosed by conventional cytology; (iii) Molecular testing provides genetic information on the inherited nature of diseases that can be directly investigated in cytology samples, by either exfoliation or by fine needle aspiration; (iv) Sometimes the cytopathology sample is the most convenient (or the only available) source of material for molecular testing; (v). Direct molecular interrogation of cells allows for a diagnostic correlation that would otherwise not be possible. Parallel to this direct diagnostic implication, cytopathology is increasing important in the validation of biomarkers for specific diseases, and in therefore of significant importance in the overall translational research strategies. We illustrate its application in some of the main areas of oncology molecular testing, such as molecular fingerprinting of neoplasms,3 lymphoreticular diseases,2 sarcomas4 and lung cancer,5 as well as translational research using diagnostic cytopathology techniques. The next years will see the consolidation of Diagnostic Molecular Cytopathology, a process that will lead to a change of many paradigms. In general, diagnostic pathology departments will have to reorganize molecular testing to pursue a cost-efficient operation. Sample preparation will have to take into account optimal preservation of nuclear acids. The training of technical staff and the level of laboratory quality control and quality assurance would have to follow strict clinical (not research) laboratory parameters. And, most importantly, those pathologists undertaking molecular diagnosis as a discipline would have to develop their professional expertise within the same framework of fellowships and professional credentials that is offered in other sub-specialties. The price to pay if this effort is not undertaken is too important for the future of diagnostic pathology in general. The increasing characterization of molecular biomarkers with diagnostic, prognostic or therapeutic value is making the analysis of tissue and cell samples prior to treatment a more complex exercise. If cytopathologists and histopathologists allow others to take charge of molecular diagnosis, our overall contribution to the diagnostic process will be diminished. We may not become less important, but we may become less relevant. However, those within the discipline of diagnostic pathology who can combine the clinical background of diseases with the morphological, immunocytochemical and molecular diagnostic interpretation will represent bona fide diagnostic specialists. Such ,molecular cytopathologists' would place themselves at the centre of clinical decision-making. Reference:, 1. Liz Fletcher. Roche leads molecular diagnostics charge. Nature Biotechnol 20, 6,7; 2002 2. Salto-Tellez M and Koay ESC. Molecular Diagnostic Cytopathology - Definitions, Scope and Clinical Utility. Cytopathology 2004; 15:252,255 3. Salto-Tellez M, Zhang D, Chiu LL, Wang SC, Nilsson B, and Koay ESC. Immunocytochemistry Versus Molecular Fingerprinting of Metastases. Cytopathology, 2003 Aug; 14(4):186,90. 4. Chiu LL, Koay SCE, Chan NL and Salto-Tellez M. Molecular Cytopathology: Sequencing of the EWS-WT1 Gene Fusion Transcript in the Peritoneal Effusion of a Patient with Desmoplastic Small Round Cell Tumour. Diagnostic Cytopathology, 2003 Dec; 29(6): 341,3. 5. TM Chin, D Anuar, R Soo, M Salto-Tellez, WQ Li, B Ahmad, SC Lee, BC Goh, K Kawakami, A Segal, B Iacopetta, R Soong. Sensitive and Cost-Effective deptection of epidermal growth factor Receptor Mutations in Small Biopsies by denaturing High Performance Liquid Chromatography. (In press). [source] Significance of bacterial identification by molecular biology methodsENDODONTIC TOPICS, Issue 1 2004David A. Spratt Rapid advances in molecular biology over the last 20 years have provided a bewildering array of techniques aimed at helping us to tease apart all aspects of biology. The discipline of microbiology has gained greatly from these advances especially with respect to detection and identification of micro-organisms. Indeed these molecular biology techniques have changed the way we classy all life on Earth. An important part of endodontic microbiology is detection and identification of the micro-organisms associated with initiation and progression of this polymicrobial infection. A range of appropriate molecular techniques are reviewed in the present article and include aspects of comparative 16S rRNA gene sequencing, polymerase chain reaction detection, strategies for identification of unculturable bacteria, and whole community analysis. Some of these techniques are widely used in endodontic microbiology while others are used by only a few workers. The advantages and disadvantages of all the techniques are discussed and put into perspective. [source] Multiple species of the dinophagous dinoflagellate genus Amoebophrya infect the same host speciesENVIRONMENTAL MICROBIOLOGY, Issue 11 2003Paulo S. Salomon Summary Populations of the dinoflagellate Dinophysis norvegica in the Baltic Sea and in the adjacent North Sea are infected by the endoparasite Amoebophrya sp. The high diversity recently unveiled within the genus Amoebophrya brings uncertainty about their identities. We applied molecular biology techniques , 18S rDNA sequencing and fluorescent in situ hybridization (FISH) , to compare this host,parasite system from both environments. The North Sea Amoebophrya sp. 18S rDNA sequence was 89% identical to the previously described Baltic Sea Amoebophrya sp. sequence, suggesting they are different species. In spite of that, a phylogenetical analysis placed the North Sea parasite sequence in a well-supported cluster with other Amoebophrya sp. sequences. The D. norvegica 18S rDNA sequences from both environments were 100% identical, indicating that the hosts have not evolved independently. A DNA probe designed for the Baltic Sea Amoebophrya sp. 18S rRNA was used in FISH assays on infected D. norvegica populations from both environments. The probe stained all infected cells from the Baltic sample, whereas none from the North Sea were stained. The results indicate that D. norvegica is released from one parasite when entering the Baltic Sea, and become less infected by an alternative parasite species. [source] Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitroEXPERIMENTAL DERMATOLOGY, Issue 4 2007Mónika Gönczi Abstract:, Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of ,27 ± 4 to ,51 ± 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases. [source] Gastrin suppresses the interdependent expression of p16 and anion exchanger 1 favoring growth inhibition of gastric cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 6 2010Hua Tian Abstract Our previous studies demonstrated that expression and interaction of p16 with anion exchanger 1 (AE1) in gastric cancer cells is correlated with progression and shorter survival of the cancer. In this article, the effects of gastrin on p16 and AE1 and its implication in prevention and treatment of gastric cancer were studied by molecular biology techniques, animal experiment and clinical analysis. The results showed that expression of p16 in human gastric body carcinoma was downregulated along with the progression of the cancer, suggesting the reverse correlations between gastrin and p16 in vivo. Further experiments indicated that gastrin suppressed the expression of p16 via the p16 promoter and thereafter resulted in the degradation of AE1 in gastric cancer cells. Silencing of AE1 or p16 significantly inhibited the proliferation of the cancer cells. Using a xenograft tumor model in nude mice, we showed that experimental systemic hypergastrinemia induced by the administration of omeprazole led to decreased expression of AE1 and p16 as well as to a marked growth inhibition of SGC7901 tumors. It is concluded that a moderate plasma gastrin level is beneficial to the growth inhibition of gastric cancer by suppressing the expression of AE1 and p16. This finding may have an important implication for the prevention and treatment of cancers arise in the gastric antrum. [source] Diagnosis of tuberculosis: Available technologies, limitations, and possibilitiesJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2003Sanjay K. Garg Abstract Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis. However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives. Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods. Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests. There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis. A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful. It has been difficult to develop an ELISA utilizing a suitable antigen because M. tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic. With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis. The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection. However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination. In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections. J. Clin. Lab. Anal. 17: 155,163, 2003. © 2003 Wiley-Liss, Inc. [source] The M5 muscarinic receptor as possible target for treatment of drug abuseJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 6 2009R. B. Raffa Summary Two reports published in the latter 1980s are generally given credit for being the first to announce the discovery of a new subtype of muscarinic acetylcholine receptor (mAChR), designated m5 or M5, and now officially M5 (1). Both identifications were assigned using molecular biology techniques. Then , as now , no selective high-affinity ligands or toxins were available. In situ hybridization and reverse-transcriptase PCR have found M5 AChR expression in brain to be distinct from that of the four other G protein-coupled mAChR subtypes and primarily localized to the substantia nigra, ventral tegmental area, hippocampus (CA1 and CA2 subfields), cerebral cortex (outermost layer) and striatum (caudate putamen). M5 AChR brain region localization and involvement in the regulation of striatal dopamine release and in rewarding brain stimulation suggests a possible role for M5 AChR as a target for novel therapy to treat excess hedonic drive, including drug abuse. [source] REVIEW: Vitamin transport and homeostasis in mammalian brain: focus on Vitamins B and EJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Reynold Spector Abstract With the application of genetic and molecular biology techniques, there has been substantial progress in understanding how vitamins are transferred across the mammalian blood,brain barrier and choroid plexus into brain and CSF and how vitamin homeostasis in brain is achieved. In most cases (with the exception of the sodium-dependent multivitamin transporter for biotin, pantothenic acid, and lipoic acid), the vitamins are transported by separate carriers through the blood,brain barrier or choroid plexus. Then the vitamins are accumulated by brain cells by separate, specialized systems. This review focuses on six vitamins (B1, B3, B6, pantothenic acid, biotin, and E) and the newer genetic information including relevant ,knockdown' or ,knockout' models in mice and humans. The overall objective is to integrate this newer information with previous physiological and biochemical observations to achieve a better understanding of vitamin transport and homeostasis in brain. This is especially important in view of the newly described non-cofactor vitamin roles in brain (e.g. of B1, B3, B6, and E) and the potential roles of vitamins in the therapy of brain disorders. [source] Pathogenesis of Brain and Spinal Cord Atrophy in Multiple SclerosisJOURNAL OF NEUROIMAGING, Issue 2004Alireza Minagar MD ABSTRACT For more than a century, multiple sclerosis was viewed as a disease process characterized by oligodendrocyte and myelin loss, and research into the pathogenesis of multiple sclerosis was mainly focused on the mechanisms of inflammation. However, with development of more sophisticated neuroimaging and molecular biology techniques, attention has shifted to new aspects of pathogenesis of multiple sclerosis: axonal loss and neurodegeneration. Evidence is increasing that tissue destruction, primarily axonal loss and neurodegeneration, is a key element in the pathogenesis of multiple sclerosis. In addition, it is now known that brain and spinal cord atrophy begins early in the disease process of multiple sclerosis and advances relentlessly throughout the course of the disease. Cumulative data suggest that axonal loss is the major determinant of progressive neuro logic disability in patients with multiple sclerosis. Magnetic resonance imaging and magnetic resonance spectroscopy in patients with multiple sclerosis for < 5 years indicate brain atrophy and loss of axonal integrity. Neurodegeneration and axonal loss in patients with multiple sclerosis are initially accompanied by a local response from oligodendrocyte progenitor cells and some remyelination. However, these repair mechanisms eventually fail, and patients typically develop generalized brain atrophy, cognitive decline, and permanent disability. Although the exact mechanisms underlying central nervous system atrophy in patients with multiple sclerosis are largely unknown, evidence exists that atrophy may represent an epiphenomenon related to the effects of dynamic inflammation within the central nervous system, including demyelination, axonal injury, neuronal loss, Wallerian degeneration, and possibly iron deposition. This article summarizes the potential mechanisms involved in central nervous system atrophy in patients with multiple sclerosis. [source] Orthodontically stressed periodontium of transgenic mouse as a model for studying mechanical response in bone: The effect on the number of osteoblastsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 2 2000Dubravko Pavlin A better understanding of cellular and molecular mechanisms involved in response to mechanical stress is a prerequisite for future improvements in orthodontic treatment. To expand the application of molecular biology techniques in this area of research, we developed and characterized a mouse tooth movement model. The aim of this study was to biomechanically characterize this model and to evaluate the effect of orthodontic stress on the proliferation of periodontal osteoblasts. We used an orthodontic coil spring appliance with a low force/deflection rate, which produced an average force of 10,12 g. This design provided a predictable tipping movement of the molar with the center of rotation at the level of root apices. Histological observations of paradental tissues revealed a response favoring a fast onset of tooth movement and deposition of new osteoid starting after 3 days of treatment. The effect of treatment on the histomorpometric parameter of the number of osteoclasts per unit bone perimeter was determined after 1, 2, 3, 4, 6, and 12 days of treatment. Starting with day 2, the osteoblast number showed a modest but consistent increase in treated periodontal sites at all time-points, ranging from 14 to 39% and becoming significant only at day 6. Only a moderate increase in the number of osteoblasts in the areas of otherwise intense bone matrix synthesis suggests that, during bone formation, proliferation of cells has a smaller role compared to a marked increase in differentiation of individual cells. The mouse model, which allows for a controlled, reproducible, orthodontic mechanical loading, can be applied to both wild-type and transgenic animals and should enhance the research of the transduction of mechanical orthodontic signal into a biological response. [source] Molecular Tools to Study Physcomitrella patensPLANT BIOLOGY, Issue 3 2005W. Frank Abstract: The moss Physcomitrella patens has become a suitable model plant system for the analysis of diverse aspects of modern plant biology. The research strategies have been influenced by the implementation of state-of-the-art cell culture and molecular biology techniques. The forthcoming completion of the Physcomitrella genome sequencing project will generate many open questions, the examination of which will rely on a diverse set of molecular tools. Within this article, we intend to introduce the essential cell culture and molecular biology techniques which have been adopted in recent years to make Physcomitrella amenable to a wide range of genetic analyses. Many research groups have made valuable contributions to improve the methodology for the study of Physcomitrella. We would like to apologise to all colleagues whose important contributions could not be cited within this manuscript. [source] Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH),APMIS, Issue 11-12 2004BIRGITTA SCHWEICKERT This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2). [source] A research project-based and self-determined teaching system of molecular biology techniques for undergraduates,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 3 2008Shuping Zhang Abstract Molecular biology techniques play a very important role in understanding the biological activity. Students who major in biology should know not only how to perform experiments, but also the reasons for performing them. Having the concept of conducting research by integrating various techniques is especially important. This paper introduces a research project-based and self-determined teaching system of molecular biology techniques for undergraduates. Its aim is to create an environment mimicking real research programs and to help students build up confidence in their research skills. The students are allowed to explore a set of commonly used molecular biology techniques to solve some fundamental problems about genes on their own. They find a gene of interest, write a mini-proposal, and give an oral presentation. This course provides students a foundation before entering the research laboratory and allows them to adapt easily to real research programs. [source] Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc-93 gene using PCRBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 3 2005James L. Lissemore Abstract PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the nematode Caenorhabditis elegans and use PCR to detect deletions in the C. elegans unc-93 gene. In advance of the exercises, wild-type and three different unc-93 deletion mutant strains are grown, harvested, and frozen by the instructor. In one approach, students isolate genomic DNA from each strain using a genomic DNA isolation kit and use agarose gel electrophoresis to analyze the DNA and to estimate its concentration. PCRs using primers directed to two different regions of the unc-93 gene are carried out on the genomic DNA from wild-type and mutant strains, and the PCR products are analyzed by agarose gel electrophoresis. Students analyze the gel to determine the approximate location and size of deletions in the three mutant strains. Alternatively, students may lyse single nematodes and carry out PCR in one laboratory session. These exercises should be easily adaptable to detection of well characterized deletions in any organism. [source] Crime scene investigation: An exercise in generating and analyzing DNA evidence,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2003Karen M. Lounsbury Abstract The goal of this project is to introduce students to molecular biology techniques using an experimental setting that inspires both scientific and personal interest. The project is designed as a small group apprenticeship for gifted high school juniors or seniors who can spend full time in a sponsor's laboratory for at least 1 week. The students begin by examining evidence from a mock crime scene that consists of hair samples from the crime scene and from five potential suspects. Students extract DNA from the hair samples and amplify a hypervariable region within the mitochondrial genome using the polymerase chain reaction. Amplified products are then sequenced and compared with the crime scene sequence using DNA alignment software. In consecutive projects, students from four different schools successfully identified the suspect who matched the crime scene evidence. This project is a valuable learning tool not only due to the comprehensive introduction to molecular biology techniques but also because it helps the students to connect scientific exploration with well publicized media events and provides a window into potential career opportunities in the field of molecular biology. [source] A perspective of metabolic engineering strategies: moving up the systems hierarchyBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2003Thomas Bulter Abstract Metabolic engineering has been established as an important field in biotechnology. It involves the analysis, design, and alteration of the stoichiometric network using sophisticated mathematical and molecular biology techniques. It allows for improvement of pathway kinetics by removing flux bottlenecks, balancing precursors, and recycling cofactors used to increase product formation. The next step in the systems hierarchy is the constructive manipulation of regulatory networks. As our understanding of regulation continues to expand rapidly, engineering of intracellular regulation will become an integral aspect of metabolic engineering. © 2003 Wiley Periodicals, Inc. [source] | |||||||||||||