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PubMed2002

[DNA chip technology and its application in toxicology [correction of toxiology]].

DNA chip is a new molecular biology technology rapidly developed in recent years. It fixes thousands of oligonucleotide on a silicon chip of 1 cm2, hybridizes the measuring materials marked with fluorescein or isotope with probe in DNA chip, and gets the fluorescent signal of hybridized probe through the scan of confocalmicroscope. This technology has been extensively used in HGP, DNA sequencing, transcript analysis, gene diagnosis etc. Toxicology is an important branch of life science. DNA chip can be extensively used in the toxicological [correction of toxiological] field. This paper reviewed the DNA chip technology in mechanism, manufacturing method, signal check, advantages and application prospect in toxicology.

Aerospace Medicine↗
PubMedDate not supplied

Subviral pathogens of plants: the viroids.

Research during the last 15 years has conclusively shown that viroids are not only fundamentally different from viruses at the molecular level, but that they are most likely not directly related to viruses in an evolutionary sense. Today, viroids are among the most thoroughly studied biological macromolecules. Their molecular structures have been elucidated to a large extent, but much needs to be learned regarding the correlation between molecular structure and biological function. The availability of the tools of recombinant DNA technology in viroid research promises rapid progress in these areas of inquiry.

Plant Diseases↗
PubMed2005

Identification of Mytilus spp. and Pecten maximus in Irish waters by standard PCR of the 18S rDNA gene and multiplex PCR of the 16S rDNA gene.

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.

Animals↗
PubMed1988

The Annotator's Assistant: an expert system for direct submission of genetic sequence data.

As DNA sequencing technology improves and more rapid techniques become routine in molecular biology labs, researchers need to expedite the incorporation of information into genetic sequence databases, such as GenBank, by directly submitting sequence data. The Annotator's Assistant is an expert system that runs on an IBM PC and helps the molecular biologist, who may have little knowledge of the structure or content of a GenBank entry, to construct a complete and valid sequence submission file. This expert system uses a simple molecular biology knowledge base and a selection of customized screen entry forms to guide the user through the entry and annotation of a sequence and its biological features. The system compiles information about the contributor, journal references, physical and functional characteristics of the nucleic acid, source organism and features, and checks it to eliminate incomplete answers and simple errors. Users supply input by answering direct and multiple-choice questions, selecting menu items and completing entry forms; on-line help is available. Users may also enter new or unusual information using generic forms. Several modules of the expert system were converted into Prolog programs and compiled, decreasing the running time significantly. The expert system rules and the data entry forms are easy to modify, update and customize for specific sequence classes.

Base Sequence↗
PubMed1990

High speed DNA sequencing by capillary electrophoresis.

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.

Base Sequence↗
PubMed1990

Ethics and the new genetics.

Recent developments in recombinant DNA technology have resulted in a rapidly expanding range of new diagnostic and therapeutic opportunities for families with inherited disorders. They are also beginning to impinge on society as a whole as progressively more is learned about the human genome, as the community becomes more aware of the role of genes in disease, and as we start to consider the implications of being able to define individual susceptibility to both monogenic and multifactorial disorders. As we implement the new technologies, careful consideration must be given to the ethical issues which arise so that maximum benefit can be obtained by individuals and the wider community.

Ethics, Medical↗
PubMed1989

Splice of life: toward understanding genetic determinants of oral diseases.

The introduction of recombinant DNA technology has led to a rapid advancement of our knowledge of genes and genomic structure. Such technology, applied to the human genome, has provided valuable information concerning the nature and possible treatment of inherited disorders throughout the human life span. A number of oral diseases can be attacked by use of molecular biological techniques in conjunction with genetic linkage analysis. It will be possible to identify carriers of inherited diseases, to utilize prenatal and antenatal diagnosis when appropriate, and to pursue studies on the mode of inheritance of specific oral diseases. Present and projected advances in this dimension of dental research provide the knowledge and strategies for approaching and solving a number of major problems as we continue to pave the way for improvements in the diagnosis, treatment, and prevention of many oral and craniofacial diseases. This position paper examines a few of the promising areas and suggests future goals toward establishing a knowledge of the genetic determinants for oral diseases.

Humans↗
PubMed2004

Recombinant human follicle-stimulating hormone: a controversial issue--without controversy.

Recent developments in recombinant DNA technology have resulted in a rapidly expanding range of new diagnostic and therapeutic opportunities. This technology paved the way for identification, isolation, cloning and production of specific proteins. Recently, recombinant human gonadotropins became available for clinical use. Pharmacokinetics, receptor availability, pharmacodynamics and safety were extensively studied and the drugs were found to be identical, if not superior, to urinary gonadotropins that have been used in reproductive medicine for the last 40 years. It is currently apparent that the use of recombinant human gonadotropins is expected to provide improved batch-to-batch consistency, steady supply and most importantly, a purified compound with highly specific activity, which accounts for > 99% of the protein content of the preparation, making it possible to administer subcutaneously. The purpose of this report is to describe and evaluate recent developments in the use of recombinant human follicle-stimulating hormone.

Combinatorial Chemistry Techniques↗
PubMed2002

Pyrosequencing technology and the need for versatile solutions in molecular clinical research.

An increasing amount of genetic information is rapidly becoming available to the practitioners of medicine and pharmacology. This knowledge promises to revolutionize the determination of diagnoses and prognoses for genetically-based disorders as well as infectious diseases and to enable tailoring of treatment to suit the individual patient. As genomics becomes ripe for clinical implementation, versatile technologies that can handle all the relevant types of analyses will be requested by many clinicians. The recently established Pyrosequencing technology for rapid determination of short DNA sequences has gained widespread acceptance and is being used in a broad range of applications. It can provide a solution for many emerging issues in molecular clinical research and applications, owing to its reliability and high flexibility together with its user-friendly attributes.

Biomedical Research↗
PubMed2005

[Cloning and expressing of cellulase gene (cbh2) from thermophilic fungi Chaetomium thermophilum CT2].

Chaetomium thermophilum CT2 can produce extracellular cellulase with industrial value. We designed two degenerate primers to amplify catalytic domain sequence of cellobiohydrolase II ( CBH II). Full length of cDNA was obtained by rapid amplification of cDNA ends technologies. DNA sequencing revealed that cbh2 has an open reading frame of 1428bp, which encodes a putative polypeptide of 476 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 53 kD and the cbh2 consists of a fungal-type carbohydrate binding domain (CBD) separated from a catalytic domain by a linker region rich in proline/serine/threonine. PCR product consisting of the entire CBH II coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. Highly efficient production of the cellobiohydrolase II was achieved in P. pastoris under the control of the AOX1 promoter, and the expressing level was 1.2 mg/mL by small-scale culturing. The recombinant cellobiohydrolase II was purified by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography. A molecular mass of the purified enzyme is 67 kD determined by SDS-PAGE and this is similar to the native cellobiohydrolase II purified from C. thermophilum CT2. The recombinant enzyme exhibited optimum catalytic activity at pH 4.0 and 50 degrees C respectively. It was thermostable at 50 degrees C and retained 50% of its original activity after 30 min at 70 d degrees C . The high level of fully active recombinant cellobiohydrolase II got from P. pastoris makes this expression system attractive for fermentor and industrial applications.

Amino Acid Sequence↗
PubMed1985

The attitudes of religious, environmental, and science policy leaders toward biotechnology.

Biotechnology is an increasingly visible and important item on the national science policy agenda. With growing corporate and governmental funding, basic research in recombinant DNA and related technologies is expanding rapidly. In a parallel and related process, new agricultural, medical, and other applications are being developed and a growing list of genetically-engineered products is ready for field testing and market distribution. In the months and years ahead, the flow of genetically-engineered products into the marketplace and the media coverage related to those new products will increase the public's awareness of biotechnology. In addition, it is possible that some public policy debate will occur over the issue of field testing new genetically-engineered materials. Media coverage of this type of controversy will also heighten awareness and influence the aggregate level of public awareness of biotechnology. The prospect of a public debate or controversy over any biotechnology issue illustrates a fundamental problem in the formulation of science policy within a democratic political system. The processes and techniques involved in genetic engineering and complex and require some level of scientific background knowledge. A 1979 study found that only seven per cent of American adults met a minimal definition of scientific literacy. It is clear that the current levels of public awareness and knowledge about biotechnology will not allow a public policy debate similar to those associated with controversies involving Social Security or gasoline prices. How, then, does a democratic society establish public policies on advanced technical issues like biotechnology? This report will outline a model of policy formulation for specialized issues and describe the results of a national study of relevant policy leaders concerning biotechnology. To understand the formulation of public policy toward biotechnology, it is necessary to focus on the role of the policy leaders in a stratified model. In general, when there is agreement on a given policy between the decision-makers and the policy leaders, the policy is implemented and there is no wider involvement in the policy formulation process. A large number of science and technology policy issues have been handled in this manner in the decades since the Second World War and it is likely that most science policy matters will continue to be resolved directly between policy leaders and decision-makers. Looking at the general science policy formulation process, it is clear that policy leaders are an important component.(ABSTRACT TRUNCATED AT 400 WORDS)

Attitude↗
PubMed2000

Application of forensic DNA testing in the legal system.

DNA technology has taken an irreplaceable position in the field of the forensic sciences. Since 1985, when Peter Gill and Alex Jeffreys first applied DNA technology to forensic problems, to the present, more than 50,000 cases worldwide have been solved through the use of DNA based technology. Although the development of DNA typing in forensic science has been extremely rapid, today we are witnessing a new era of DNA technology including automation and miniaturization. In forensic science, DNA analysis has become "the new form of scientific evidence" and has come under public scrutiny and the demand to show competence. More and more courts admit the DNA based evidence. We believe that in the near future this technology will be generally accepted in the legal system. There are two main applications of DNA analysis in forensic medicine: criminal investigation and paternity testing. In this article we present background information on DNA, human genetics, and the application of DNA analysis to legal problems, as well as the commonly applied respective mathematics.

DNA↗
PubMed2025

A Comprehensive Bioinformatics Approach to Analysis of Variants: Variant Calling, Annotation, and Prioritization.

Next-Generation Sequencing (NGS), also known as high-throughput sequencing technologies, has enabled rapid and efficient sequencing of large amounts of DNA and RNA. These technologies have revolutionized the field of genomics, transcriptomics, and proteomics and have been widely used in cancer research, leading to advances in clinical diagnosis and treatment. Improvements in the NGS technologies enabled millions of fragments to be sequenced simultaneously in a time- and cost-effective manner and resulted in large amount of genomic data which require efficient analysis methods. Analysis of the genomic data requires both efficient computer resources and bioinformatics approaches. This chapter details a comprehensive computational approach and analysis steps for genomic data analysis.

Computational Biology↗
PubMed2002

Ultra-rapid DNA analysis using HyBeacon probes and direct PCR amplification from saliva.

We describe a novel probe technology, termed HyBeacons, which provides a new homogeneous method for fluorescence-based sequence detection and allele discrimination. Employing a single nucleotide polymorphism located in the N-acetyltransferase 2 gene as a model system, we demonstrate the utility of HyBeacon probes for rapid and reliable sequence analysis. We also demonstrate that homozygous and heterozygous samples may be accurately identified using a single HyBeacon oligonucleotide. Polymorphic DNA sequences were detected and differentiated by real-time PCR and melt peak methodologies, without performing extraction of genomic DNA prior to target amplification. Employing a combination of homogeneous HyBeacon analysis, the rapid thermal cycling conditions of the LightCycler and direct amplification from saliva, allowed samples to be genotyped within 30 min. Such rapid non-invasive diagnostic technologies may permit 'point-of-care' genetic testing to be performed in hospitals and doctor's surgeries.

Arylamine N-Acetyltransferase↗
PubMed1986

A Legionella-specific DNA probe detects organisms in lung tissue homogenates from intranasally inoculated mice.

We have prepared a DNA probe from internal sequences of the gene encoding the Legionella pneumophila major outer membrane protein (MOMP). Immunologic studies of the MOMP have confirmed that it possesses both genus-specific and species-specific antigenic domains, but possesses no cross-reactivity with non-Legionella species. At the DNA levels, the 3' half of the gene contains sequences that are homologous to DNA from all strains tested within the genus, whereas the 5' half of the gene has homology with L. pneumophila strains only. Homology of the gene with non-legionellae has not been detected even under low stringency conditions. To test the utility of this probe for detecting organisms in tissue, we tested crude homogenates of mouse lungs representing 1/1,000th of the total lung mass. After intranasal inoculation with 2 X 10(8) colony-forming units of L. pneumophila, mice were sacrificed at various intervals (10 mice per group). Since L. pneumophila does not produce a propagating infection in these animals, cultures of lung tissue from successive days after inoculation showed a roughly linear decline in viable L. pneumophila (total lung yield: 10(8) on Day 0, 5 X 10(7) on Day 2, 10(5) on Day 5, 10(3) on Day 9, and less than 10(2) on Day 15). By DNA dot hybridization with the MOMP probe, we detected positive signals from most animals on Days 0 and 2, suggesting a threshold sensitivity of between 50,000 and 100,000 organisms with our current methods. Advances in DNA probe technology may soon permit the rapid, specific identification of either L. pneumophila or other Legionella species in pathologic specimens.

Animals↗
PubMed2002

Options available--from start to finish--for obtaining data from DNA microarrays II.

Microarray technology has undergone a rapid evolution. With widespread interest in large-scale genomic research, an abundance of equipment and reagents have now become available and affordable to a large cross section of the scientific community. As protocols become more refined, careful investigators are able to obtain good quality microarray data quickly. In most recent times, however, perhaps one of the biggest obstacles researchers face is not the manufacture and use of microarrays at the bench, but storage and analysis of the array data. This review discusses the most recent equipment, reagents and protocols available to the researcher, as well as describing data analysis and storage options available from the evolving field of microarray informatics.

DNA↗
PubMed2002

Microarray analysis of gene expression changes in aging in mouse submandibular gland.

Little is known about the effect of salivary gland function during aging based on gene expression. Recently emerged DNA array technology provides a sensitive, quantitative, rapid approach to the monitoring of the global pattern of gene expression. In this study, we used high-density oligonucleotide arrays to monitor the changes of gene expression levels in the submandibular gland (SMG) by comparing adult mice with elderly adult mice. Of the 1328 genes screened, 160 genes (12.0%) showed more than two-fold changes; 154 (96.3%) of these genes, associated with transcription regulation, transport, signal transduction, and enzymes in the elderly mice, exhibited decreased expression levels. The remaining 6 genes (3.7%) in the elderly mice showed increased expression levels. In mouse SMG, analysis of these data suggests that aging may lead the gene expression to decrease than increase. Thus, DNA array technology can be a powerful tool for the identification of age-associated candidate genes for further analysis in aging.

1-Phosphatidylinositol 4-Kinase↗
PubMed1999

Application of DNA arrays to toxicology.

DNA array technology makes it possible to rapidly genotype individuals or quantify the expression of thousands of genes on a single filter or glass slide, and holds enormous potential in toxicologic applications. This potential led to a U.S. Environmental Protection Agency-sponsored workshop titled "Application of Microarrays to Toxicology" on 7-8 January 1999 in Research Triangle Park, North Carolina. In addition to providing state-of-the-art information on the application of DNA or gene microarrays, the workshop catalyzed the formation of several collaborations, committees, and user's groups throughout the Research Triangle Park area and beyond. Potential application of microarrays to toxicologic research and risk assessment include genome-wide expression analyses to identify gene-expression networks and toxicant-specific signatures that can be used to define mode of action, for exposure assessment, and for environmental monitoring. Arrays may also prove useful for monitoring genetic variability and its relationship to toxicant susceptibility in human populations.

Environmental Exposure↗