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PubMed1999

Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation.

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.

Animals↗

PubMed2001

High-throughput microarray technologies: from genomics to clinics.

BACKGROUND: Despite continuous research efforts in the past decades, there are still cancers where no effective treatment is available, such as advanced kidney cancer or hormone-refractory prostate cancer. A better understanding of the molecular mechanisms of cancer development and progression is the basis for the development of new diagnostic and therapeutic strategies. Current developments in genomics have a dramatic impact on the whole field of research. The sequence of the entire human genome will soon be fully sequenced and provide the 'book of life' as a basis for the understanding of human disease. METHODS AND RESULTS: New technologies have emerged to translate the human genome sequence into gene function and improved diagnostics or treatment modalities. New technologies such as microarrays are not only important for fundamental research, but will also be useful for diagnostic, prognostic or therapeutic purposes in individual patients. DNA microarrays make it possible to analyze the mRNA expression of thousands of genes simultaneously. The resulting comprehensive gene expression surveys lead to the identification of new genes and pathways with importance in cancer development and progression, or as targets for new therapies. The validation and prioritization of genes emerging from genome screening analyses in large series of clinical tumors has become a new bottleneck in research. Therefore, we have recently developed the tissue microarray (TMA) technology to efficiently test the clinical relevance of candidate genes. TMAs are microscope slides containing samples from hundreds of individual tumor specimens. They can be used for large-scale, massively parallel in situ analysis of genetic alterations on a DNA, RNA and protein level using in situ hybridization or immunohistochemistry on hundreds of tumor specimens at a time. Microarray technologies are already increasingly being used in urologic research, and will also have a strong impact on clinical urology. CONCLUSIONS: DNA microarrays and TMAs provide a powerful approach to identify large numbers of new candidate genes, and rapidly validate their clinical impact in large series of human tumors. These technologies will soon lead to a better molecular understanding of urologic tumors, and accelerate the identification of new prognostic markers or therapeutic targets.

Clinical Medicine↗

PubMed2004

Rapid quantification of the heteroplasmy of mutant mitochondrial DNAs in Leber's hereditary optic neuropathy using the Invader technology.

PURPOSE: To quantify the degree of heteroplasmy of a mitochondrial DNA (mtDNA) mutation in Leber's hereditary optic neuropathy (LHON) a biplex Invader assay was applied. METHODS: To determine the optimum condition for the Invader assay, mtDNAs were assayed in various amounts of total DNA in 1-4-h incubations at 63 degrees C. To evaluate the suitability of the Invader assay to detect the three mutations, G3460A, G11778A, and T14484C, 10 ng of DNAs from 224 patients with bilateral optic atrophy was assayed. To quantify mtDNA heteroplasmy, a standard curve of known mixture ratios of mutation against calculation by the Invader assay was constructed. Seventy-two of the 224 patients had one of the three mutations, which corresponded with the mutation detected earlier by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. The percentages of mutant mtDNAs were calculated by the Invader assay in five heteroplasmic families, including 30 individuals with the G11778A mutation. The results were compared with those calculated earlier by labeled polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis. RESULTS: In 1-8 ng of DNA, the fluorescence intensity increased near linearly during a 4-h assay. With more than 16 ng of DNA, the intensities were saturated even at the 2-h assay. A linear relationship was observed between the results obtained from separate mixtures and from the Invader assay analysis. Because two fluorescent intensities are not always the same, one of the two intensities was modified to adjust to that of the other. Complete concordance was observed between PCR-RFLP analysis and Invader assay genotyping for the 224 patients. Results of percentage of heteroplasmy in five LHON families obtained by the Invader assay were consistent with those by the PCR-SSCP analysis. CONCLUSIONS: Invader assay is a simple, rapid, and reliable method of genotyping mtDNA mutations as well as quantifying heteroplasmy simultaneously under optimum conditions.

Base Sequence↗

PubMed1999

AFLPs represent highly repetitive sequences in Asparagus officinalis L.

The chromosomal and genomic organization of 5 cloned AFLP fragments in asparagus (Asparagus officinalis L.) were investigated. Two of the 5 AFLP loci were sex-linked. The fragments, amplified with EcoRI/Mse I primers, ranged from 107 to 267 bp and were AT-rich. Southern hybridization gave interspersed, middle repetitive to high copy sequence signals. Fluorescence in-situ hybridization (FISH) exhibited hybridization signals on all chromosomes with dispersed distribution pattern and varying signal intensities. Repetitive signals in the form of clusters were observed on all chromosomes. In addition, the 5S rRNA gene was physically mapped on one pair of chromosomes and the 18S-5.8S-25S rRNA genes on three pairs. The results of the FISH and Southern analyses showed that the AFLP marker technology relies on repetitive sequences. Since repetitive DNA sequences represent a fraction of the plant genome undergoing rapid changes during the course of evolution, the question of whether such molecular markers originating from repetitive DNA sequences remain stable is discussed.

DNA Probes↗

PubMed2003

[Molecular mechanisms in muscle adaptation].

Skeletal muscle shows a unique ability to adapt its structure and function to various stimuli. The recent use of molecular-biological technology indicates that in human skeletal muscle exercise stimuli cause a rapid increase in the transcription of DNA sequences for metabolic and regulatory genes into messenger RNA. Additionally, repetitive and systematic exercise stimuli cause a change in the concentration of certain mitochondrial and contractile RNAs. These distinct changes in gene expression reflect the specific adaptations of the mitochondrial and myofibrillar structure of skeletal muscle to different stimuli as endurance exercise, strength training, and immobilization, respectively. Multiple molecular sensors have been identified which are involved in the sensation, transmission and integration of mechanical, metabolic, hormonal and neuronal signals of the more complex stimuli of exercise training into the observed adaptations of gene expression. The data support the fundamental concept, that by instructing the synthesis of encoded protein(s) changes in gene expression are responsible for the microadaptations of regularly exercised human skeletal muscles leading towards a new training steady-state.

Animals↗

PubMed1991

Rapid diagnosis of Legionella infection by a nonisotopic in situ hybridization method.

The authors report a nonradioactive adaptation of DNA hybridization technology for the direct detection of Legionella organisms in situ in routinely processed histologic specimens. The probe used consisted of synthetic oligodeoxynucleotides, complementary to the ribosomal RNA of all clinically relevant Legionella species, labeled with biotinylated dUTP at their 3' ends. By in situ DNA hybridization and detection with an avidin-alkaline phosphatase complex. Legionella was visualized by light microscopy within the alveoli of lung specimens in 9 of 13 direct fluorescent antibody- or culture-positive cases of Legionnaires' disease. No cross-hybridization was observed in lung specimens infected with Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or other pathogens. The authors' results illustrate a novel adaptation of in situ DNA hybridization techniques, usually used for viruses, to the detection of a bacterial organism. The method enables direct visualization of bacterial nucleic acid in infected tissues and may facilitate early diagnosis and treatment of legionellosis.

Adult↗

PubMed1999

Fast-in situ hybridization and immunoenzymatic color pigment detection of mouse bone marrow micronucleus.

The development of a whole mouse genomic DNA probe coupled to color pigment painting detection methodology can accurately verify mouse micronuclei induced by chemicals or drugs leading to a lower probability of potential artifacts. Using color pigment painting detection of probes in conjunction with Wright's Giemsa counterstain instead of the current fluorescence detection technology ensures low cost, high resolution permanent documentation of slides for a particular test compound. The permanent color pigment-detected micronuclei and adjoining counterstain allows slides to be stored for future analysis without enhancing the signal or adding antifading agents that are associated with fluorescence detection. Combining innovative technology such as fast-in situ hybridization of DNA probes with immunoenzymatic color pigment detection provides rapid verification of true micronuclei (DNA containing) within 2-3 hr.

Animals↗

PubMed1990

[Current trends in parasitological diagnosis].

During the past few years a number of modern techniques has found its way into the discipline of diagnostic parasitology. The scale of these new tool ranges from highly sensitive and low toxic fixatives for intestinal protozoa, to rapid detection methods for malarial parasites and to the techniques of modern biology, like hybridoma and DNA technology.

Animals↗

PubMed2005

Application of FTA technology to extraction of sperm DNA from mixed body fluids containing semen.

FTA technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. In this study, we report a rapid and simple method of extracting DNA from sperm when body fluids mixed with semen were collected using FTA cards. After proteinase K digestion of the sperm and body fluid mixture, the washed pellet suspension as the sperm fraction and the concentrated supernatant as the epithelial cell fraction were respectively applied to FTA cards containing DTT. The FTA cards were dried, then directly added to a polymerase chain reaction (PCR) mix and processed by PCR. The time required from separation of the mixed fluid into sperm and epithelial origin DNA extractions was only about 2.5-3h. Furthermore, the procedure was extremely simple. It is considered that our designed DNA extraction procedure using an FTA card is available for application to routine work.

DNA↗

PubMed2003

DiscernArray technology: a cell-free method for the generation of protein arrays from PCR DNA.

Protein array technology offers a powerful tool to bridge genomics and proteomics. Currently, the bottleneck in the generation of protein arrays is the comprehensive production of functional proteins. We have developed a rapid cell-free method, DiscernArray, which creates functional protein arrays directly from PCR DNA by in vitro synthesis of individual tagged proteins on tag-binding surfaces, such that the tagged proteins are immobilized on a surface as they are synthesised. DiscernArray is particularly useful for arraying proteins and domains which cannot be functionally produced in heterologous expression systems or for which the cloned DNA is not available.

Antibody Specificity↗

PubMedDate not supplied

Nucleic acid probes in periodontal diagnosis.

Periodontal Disease is considered as an infection caused by a variety of microorganisms. Considering the vast range of microbial species involved in the causation of periodontal diseases, a specific diagnostic procedure, to identify the various organisms involved is a major necessity. A number of diagnostic procedures, including microbiological and immunological have been utilized in the diagnosis of periodontal disease. The DNA probe technology provides both a sensitive and specific assay and also alleviates concern for transport of the fastidious microorganisms in clinical samples, fulfills the need for a specific and rapid test, that does not require the preservation of viable microorganisms. DNA probes are now being used to identify the various putative pathogen's including; A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus, etc. Besides DNA probes have also been proven an advantage over the various other available procedures in periodontal diagnosis.

Aggregatibacter actinomycetemcomitans↗

PubMedDate not supplied

DNA fingerprinting. The future of forensic dentistry--a review.

Forensic dental identification is at technological cross roads. The incidence of dental restorations, the mainstay of radiographic dental investigations, have declined. Whereas molecular biology laboratory procedures are rapidly increasing in efficiency and availability. With new typing techniques forensic scientists can characterize individuals at the fundamental level of their DNA, and variations between individuals at this level can be used to discriminated between them. The anatomical location of teeth and the extent to which teeth may suffer environmental changes and still provide useful DNA material has propelled forensic odontology. The techniques of DNS fingerprinting, the role of teeth as a source of DNA material and its feasibility is discussed in relatively simple terms.

DNA↗

PubMed1998

Generation of co-dominant PCR-based markers by duplex analysis on high resolution gels.

Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high-performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready-to-use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single-nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co-dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36 Arabidopsis thaliana loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half (n = 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co-dominant PCR-based molecular markers for fine-scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.

Alleles↗

PubMed1999

Engineering baker's yeast: room for improvement.

Bread making is one of the oldest food-manufacturing processes. However, it is only in the past few years that recombinant-DNA technology has led to dramatic changes in formulation, ingredients or processing conditions. New strains of baker's yeast that produce CO2 more rapidly, are more resistant to stress or produce proteins or metabolites that can modify bread flavour, dough rheology or shelf-life are now emerging.

Cellulose↗

PubMed1991

Enzyme assays.

The past year or so has seen the development of new enzyme assays, as well as the improvement of existing ones. Assays are becoming more rapid and sensitive as a result of modifications such as amplification of the enzyme product(s). Recombinant DNA technology is now being recognized as a particularly useful tool in the search for improved assay systems.

Animals↗

PubMed2002

Protein therapeutics: promises and challenges for the 21st century.

Recent advances in massively parallel experimental and computational technologies are leading to radically new approaches to the early phases of the drug production pipeline. The revolution in DNA microarray technologies and the imminent emergence of its analogue for proteins, along with machine learning algorithms, promise rapid acceleration in the identification of potential drug targets, and in high-throughput screens for subpopulation-specific toxicity. Similarly, advances in structural genomics in conjunction with in vitro and in silico evolutionary methods will rapidly accelerate the number of lead drug candidates and substantially augment their target specificity. Taken collectively, these advances will usher in an era of predictive medicine, which will move medical practice from reactive therapy after disease onset, to proactive prevention.

Animals↗

PubMedDate not supplied

Particle-mediated gene therapy of wounds.

Gene therapy is becoming a reality, and it is a particularly attractive approach for wound healing, because the wound site is often exposed, the treatment and condition should be transient, and gene products such as growth factors and cytokines suffer from problems with bioavailability and stability. Among the techniques for gene delivery to the wound site, particle-mediated bombardment with a device called the gene gun has become an important developmental tool. This instrument has been used in numerous examples of wound gene therapy with growth factors or their receptors in the last decade. Among the advantages of particle-mediated bombardment are ease and speed of preparation of the delivery vehicle, the stability of the DNA preparation, the absence of (viral) antigens, the ability to target the projectiles to different tissue depths and areas, and the rapid shedding of both particles and DNA if they are targeted to the epidermis. Clinical application of the technology remains limited by the relatively low efficiency of the method, the potential tissue damage created by impact of the particles, and the coverage area. The gene gun can also be used to facilitate the discovery and validation of gene products as wound healing agents.

Drug Carriers↗

PubMed1995

Chorionic villus sampling.

Chorionic villus sampling (CVS) has the great advantage over mid-trimester amniocentesis of producing early results. Moreover, rapid analytic techniques have significantly reduced the waiting time between sampling and diagnosis, whereas progress in recombinant DNA technology and human gene mapping has led to an increase in the range of conditions it can detect. The role of CVS in twin pregnancy has been investigated and compared with amniocentesis. Although these techniques are equally safe, CVS should be considered the approach of choice because of a number of technical advantages, and in relation to selective fetal reduction in discordant twins. Confined placental mosaicism has been investigated and a list of chromosomes related to adverse pregnancy outcome has been compiled. Recent reports have substantially contributed to solving the controversy on the hypothetical relationship between limb reduction defects and CVS. Analysis of limb reduction defects among more than 130,000 cases reported to the World Health Organization CVS registry has been unable to find any relationship between sampling and fetal malformations, including limb reduction defects.

Chorionic Villi Sampling↗