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[DNA probes in the diagnosis of infectious diseases].

With the recent and rapid developments in recombinant DNA technology it has become much easier to use nucleic acid probes in diagnosis of infectious diseases. In this review some parameters like designing of probes, selection of tags, hybridization reaction conditions and in situ hybridization have been discussed. In addition, some examples have been reported which employ techniques of dot-blot hybridization and in situ hybridization for the diagnosis of latent cytomegalovirus infection.

Bacterial Infections↗
PubMed1997

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

Chromosomes, Human↗
PubMed2006

Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.

Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system.

DNA, Fungal↗
PubMed2005

Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology.

DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.

DNA Primers↗
PubMed2001

Designing immune responses with genetic immunization and immunostimulatory DNA sequences.

Genetic immunization or DNA vaccination represents a rapidly developing technology with new perspectives for the prevention and therapy of infectious diseases and it offers new approaches for the treatment of autoimmunity, tumors and even allergy. DNA vaccines are comprised of plasmid DNA which encodes antigen molecules directly in the transfected cells of a target organism. In contrast to protein-induced immune responses, DNA vaccines stimulate both humoral and cell-mediated immune reactions. In the present review we present a palette of unique features of genetic immunization like the effect of CpG motifs, the influence of mode and site of gene delivery and the modulation of immune responses by co-delivery of cytokines, colony stimulating factors, adhesion molecules and other stimulatory molecules. In addition, modulation of the immune response via translation, processing and presentation will be discussed, which in sum demonstrate the elegant possibilities of genetic immunization to induce tailor-made immune responses.

Animals↗
PubMed2004

Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays.

We developed a new DNA microarray-based technology, called protein binding microarrays (PBMs), that allows rapid, high-throughput characterization of the in vitro DNA binding-site sequence specificities of transcription factors in a single day. Using PBMs, we identified the DNA binding-site sequence specificities of the yeast transcription factors Abf1, Rap1 and Mig1. Comparison of these proteins' in vitro binding sites with their in vivo binding sites indicates that PBM-derived sequence specificities can accurately reflect in vivo DNA sequence specificities. In addition to previously identified targets, Abf1, Rap1 and Mig1 bound to 107, 90 and 75 putative new target intergenic regions, respectively, many of which were upstream of previously uncharacterized open reading frames. Comparative sequence analysis indicated that many of these newly identified sites are highly conserved across five sequenced sensu stricto yeast species and, therefore, are probably functional in vivo binding sites that may be used in a condition-specific manner. Similar PBM experiments should be useful in identifying new cis regulatory elements and transcriptional regulatory networks in various genomes.

Base Sequence↗
PubMed1992

Probing identity: the changing face of DNA fingerprinting.

DNA-fingerprinting technology has made a very rapid transition from being a research laboratory discovery to an applied science widely understood by, and of interest to, the general public. However, DNA fingerprinting is often portrayed as being a single generic technology, rather than a complex evolving mixture of methodologies, where specific applications demand selection of appropriate probes and techniques.

Animals↗
PubMed2001

Flow-through polymerase chain reactions in chip thermocyclers.

The miniaturization of analytical devices by micromachining technology is destined to have a major impact on medical and bioanalytical fields. To meet the current demands for rapid DNA amplification, various instruments and innovative technologies have been introduced by several groups in recent years. The development of the devices was extended in different directions and adapted to corresponding applications. In this review the development of a variety of devices and components for performing DNA amplification as well as the comparison of batch-process thermocyclers with reaction chambers and flow-through devices for different purposes are discussed. The main attention is turned to a flow device concept for thermocycling using microfabricated elements for local heat flow management, for which simulations and considerations for further improvement regarding design, material choice and applied technology were performed. The present review article mainly discusses and compares thermocycling devices for rapid thermocycling made of silicon or of silicon and glass with a short excursion to the possibility of plastic chip devices. In order to perform polymerase chain reactions (PCRs) in the microreactors, special attention must be paid to the conditions of the internal surfaces. For microchips, surface effects are generally pronounced because the surface to volume ratio increases upon miniaturization. Solutions for solving this problem are presented. We propose an overview of layouts for batch-process thermocyclers with different parallelization of reaction chambers and also of different designs of continuous flow thermocycling chips, paying particular attention to the parameters which influence the efficiency of such chip devices. Finally we point out some recent issues for applications in the field of clinical diagnostics.

Bioreactors↗
PubMed2004

Rapid detection of mutations in Wilson disease gene ATP7B by DNA strip technology.

Wilson disease leads to severe hepatic and neurological pathology resulting from cellular copper overload in the respective tissue. Although the affected gene, ATP7B, has been identified, genetic testing is challenging, time-consuming and expensive. Here we describe the development and use of a novel diagnostic test for four frequent mutations (M769V, W779X, H1069Q and P1134P-fs) found in Germany and many other countries in Europe. The test is based on multiplex polymerase chain reaction and DNA strip technology and was found to be highly sensitive and specific, as well as timely and cost-effective. We conclude that this test is a useful and reliable tool to screen Wilson disease patients and their family members for these mutations and may facilitate diagnosis in this complex disease.

Adenosine Triphosphatases↗
PubMed1998

Out on the farm with DNA vaccines.

DNA vaccination is a rapidly developing technology that offers new approaches for the prevention of disease. This technology may permit the production of new vaccines against diseases that have no current vaccine, as well as allowing the development of improved vaccines to replace existing products. We describe how DNA vaccination is being developed for use in commercial animal production, with an emphasis on viral diseases, and discuss the existing hurdles to its development and use.

Animal Diseases↗
PubMed1997

Quantification of hepatitis B virus DNA using competitive PCR and a scintillation proximity assay.

A rapid assay for the quantification of hepatitis B virus DNA in human serum was developed. The principle of the method combines competitive polymerase chain reaction (cPCR) (for the controlled amplification of hepatitis B virus DNA) and scintillation proximity assay (SPA) technology (for rapid detection and quantitation of PCR products). It also incorporates a reproducible and simple method for the preparation of serum DNA suitable for PCR amplification. The assay has a better linear dynamic range than traditional methods that use 32P to detect PCR products. It was applied to a range of hepatitis B virus (HBV) surface antigen positive (HBsAg + ) sera, and shown to be more sensitive than a commercially available HBV DNA kit.

DNA, Viral↗
PubMed2003

Genotyping and species identification of Fritillaria by DNA chips.

AIM: To investigate the genetic polymorphism of several species of Fritillaria and to develop a DNA chip for the genotyping and identification of the origin of various species of Fritillaria at molecular level. METHODS: Genomic DNA from bulbs of several Fritillaria species was extracted and the polymorphisms of the D2 and D3 regions inside the 26S rDNA gene were identified by direct sequencing. Oligonucleotide probes specific for these polymorphisms were designed and printed on the poly-lysine coated slides to prepare the DNA chip. PCR products from the Fritillaria species were labeled with fluorescence by incorporation of dye-labeled dideoxyribonucleotides and hybridized to the immobilized probes on the chip. RESULTS: The polymorphisms were used as markers for discrimination among various species. Specific oligonucleotide probes were designed and immobilized on a DNA chip. Differentiation of the various Fritillaria species was accomplished based on hybridization of fluorescent labeled PCR products with the DNA chip. CONCLUSION: The results demonstrated the reliability of using DNA chips to identify different species of Fritillaria, and the DNA chip technology can provide a rapid, high throughput tool for genotyping and quality assurance of the plant species verification.

Base Sequence↗
PubMed2002

CoreGenes: a computational tool for identifying and cataloging "core" genes in a set of small genomes.

BACKGROUND: Improvements in DNA sequencing technology and methodology have led to the rapid expansion of databases comprising DNA sequence, gene and genome data. Lower operational costs and heightened interest resulting from initial intriguing novel discoveries from genomics are also contributing to the accumulation of these data sets. A major challenge is to analyze and to mine data from these databases, especially whole genomes. There is a need for computational tools that look globally at genomes for data mining. RESULTS: CoreGenes is a global JAVA-based interactive data mining tool that identifies and catalogs a "core" set of genes from two to five small whole genomes simultaneously. CoreGenes performs hierarchical and iterative BLASTP analyses using one genome as a reference and another as a query. Subsequent query genomes are compared against each newly generated "consensus." These iterations lead to a matrix comprising related genes from this set of genomes, e. g., viruses, mitochondria and chloroplasts. Currently the software is limited to small genomes on the order of 330 kilobases or less. CONCLUSION: A computational tool CoreGenes has been developed to analyze small whole genomes globally. BLAST score-related and putatively essential "core" gene data are displayed as a table with links to GenBank for further data on the genes of interest. This web resource is available at http://pumpkins.ib3.gmu.edu:8080/CoreGenes or http://www.bif.atcc.org/CoreGenes.

Algorithms↗
PubMed1994

Molecular diagnostic techniques for viral hepatitis.

The five different viruses that cause human viral hepatitis, A, B, C, D, and E share the unique property of hepatotropism. Identification of the nucleic acid sequences of each of these viruses, coupled with the application of recombinant DNA technology, has resulted in the rapid development of molecular diagnostic techniques. Currently, molecular hybridization and polymerase chain reaction are the major diagnostic techniques for viral hepatitis. This review discusses the molecular biology of the five hepatitis viruses, the principles of molecular biological techniques, and their current and future diagnostic applications.

Algorithms↗
PubMed2003

A rapid DNA probe test compared to culture methods for identification of subgingival plaque bacteria.

There has been a significant amount of interest in developing a more rapid and cost-effective test to identify bacterial pathogens in plaque. DNA probe technology may meet both these objectives, it is more rapid and cost-effective when compared to culture methods. The purpose of this study was to compare an automated DNA probe test with classical culture methods for identifying Bacteroides forsythus and Porphyromonas gingivalis in subgingival plaque of patients with adult periodontitis. Subgingival plaque samples were collected from sites with moderate to severe periodontitis and divided into two aliquots for analysis by either DNA probe or culture methods. When the DNA probe method was compared with the culture method (gold standard), the sensitivity and specificity for B. forsythus were 92.0% (SE = 3.4%) and 50.5% (SE = 7.8%), respectively; for P. gingivalis they were 52.2% (SE = 8.7%) and 74.7% (SE = 5.9%), respectively. Detection of B. forsythus and P. gingivalis by DNA probe correlated with probing depth (P = 0.01 for B. forsythus and P = 0.03 for P. gingivalis). It was concluded the DNA probe test was comparable to culture methods in detecting B. forsythus. In addition, when compared to the culture method, a better correlation was obtained with DNA probe detection of B. forsythus or P. gingivalis and clinical parameters.

Adult↗
PubMed1996

Recombinant technique and gonadotropins production: new era in reproductive medicine.

OBJECTIVE: To review current knowledge regarding recombinant DNA technology and its safety and efficacy in relation to recombinant gonadotropin production. DATA IDENTIFICATION AND SELECTION: Studies that relate specifically to recombinant DNA technology, method of laboratory production, and the clinical aspects of using recombinant gonadotropins were identified through literature and Medline searches. RESULTS: Recent developments in recombinant DNA technology have resulted in a rapidly expanding range of new diagnostic and therapeutic opportunities. This technology paves the way to the identification, isolation, cloning, and production of specific proteins. Recently, recombinant human gonadotropins became available for clinical use. The pharmacokinetics, receptor availability, pharmacodynamics, and safety were studied extensively and the drugs were found to be identical if not superior to urinary gonadotropins that have been used in reproductive medicine for the last 30 years. It is clear today that the use of recombinant human gonadotropins is expected to provide better batch-to-batch consistency, steady supply, and most importantly, a purified compound with high specific activity, which accounts for >99% of the preparation's protein content, allowing SC administration. CONCLUSION: There is no doubt that recombinant gonadotropins produced by genetic engineering technology are here to stay and will represent an important treatment modality in various fertility disturbances.

Clinical Trials as Topic↗
PubMed1996

Rapid, non-radioactive detection of virus infection by polymerase chain reaction.

BACKGROUND: Polymerase chain reaction (PCR) diagnosis of infectious diseases, especially virus diseases, offers a very sensitive and specific technique for clinical diagnosis. However, detection systems for amplified DNA requiring radioactive probe hybridization or signal development using blot transfer or nucleotide capture require overnight incubation or specially labeled probe molecules for analysis of amplified DNA. OBJECTIVES: To place this technology in the clinical laboratory, rapid and sensitive methods are needed for the detection of amplified DNA which are applicable to the assay of multiple specimens representing many different organisms and requiring a minimum of manipulation. STUDY DESIGN: Electrophoretic separation of amplified DNA fragments, stained with the fluorescent dye SYBR Green I, and laser scanning of the gels for detection of virus-specific PCR products was compared with detection of amplified DNA by liquid hybridization with radioactive probes and gel retardation analysis of labeled probe molecules. RESULTS: Fluorescent scanning methodology was applied to the detection of cytomegalovirus (CMV), herpes simplex virus (HSV) and the human immunodeficiency virus (HIV). This method was at least 10 times more sensitive than radioactive probe hybridization in the detection of CMV-specific PCR products. This method also required less time and avoided the use of radioactivity. CONCLUSIONS: Clinical diagnosis of virus infections can be conveniently and rapidly accomplished, while avoiding the dangers of radioactive probe handling, by fluorescence staining and laser scanning of specifically amplified gene fragments. This technology is applicable to the detection of genes from many different organisms, without specially synthesized and/or labeled oligonucleotide primer or probe sequences.

Journal Article↗
PubMed1996

[Insulin analogs].

The present insulin preparations are not ideal for diabetes therapy by subcutaneous administration, subcutaneous insulin absorption is too slow to mimic the normal rapid increments of insulin in blood in response to nutrient intake. The absorption of insulin from subcutaneous tissue is influenced by many factors, among which the association state of insulin pharmaceutical form (hexameric) is the most important. This review describes the properties of insulin analogues, both rapid-acting and long-acting insulin analogues, designed by recombinant DNA technology with the aim of ameliorating absorption properties. Rapid-acting insulin analogues are characterised by one or a few amino acid substitutions to reduce intermolecular binding responsible for self-association. Because of largely reduced self-association they are absorbed substantially faster after subcutaneous injections than current regular insulin and determine lower insulin levels late after injection; thus short-acting analogues can be given with the meal and can minimise late hypoglycaemia. Long-acting insulin analogues have a slow absorption, providing constant basal insulinemic levels with lower day-to-day variation than current long-acting insulins. These properties are obtained by adding positive and/or removing negative charges thus increasing the isoelectric point of the molecule to about 7. In conclusion, insulin analogues represent an important goal in the therapy of diabetes and they can be a real opportunity for ameliorating glycaemic control.

Animals↗