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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↗
PubMed2001

Rapid DNA typing of HLA-B27 allele by real-time PCR using lightcycler technology.

For clinical diagnostic routine we developed a fast DNA typing of HLA-B27 by PCR and real-time detection using LightCycler technology. The method combines the sensitivity and specificity of PCR with the swiftness of the LightCycler system. The amplification step was performed with a primer set coding for a region in the third exon common to B*2701 to B*2705. The PCR cycles were monitored continuously using the SYBR Green I dye. Beta-globin was used as an internal control. An analysis of 32 samples with one PCR run was completed within 40 minutes. After amplification a melting curve analysis permitted the accurate identification of the PCR amplicons. The mean melting temperatures (Tm) were 90.5 degrees C and 87.3 degrees C, which are characteristic for HLA-B27 and beta-globin, respectively. A comparison of 300 samples which were typed for HLA-B27 with a conventional sequence-specific polymerase chain reaction (SSP-PCR) and with the new method demonstrated a perfect correlation (specificity 100%). In summary, the method described is fast, reliable, cost-effective and well adapted for routine laboratory testing.

Alleles↗
PubMed2001

Utilization of new technologies in drug trials and discovery.

It has become widely accepted that individual genetic variation is a prime determinant in both disease susceptibility and toxic response to therapeutic agents and xenobiotics. Emerging genetic sequence data and phenotype association studies are expected to enable disease risk prediction and guide subsequent therapeutic approaches in individual cases. However, making a good match between an individual genetic profile, disease risk prediction, and appropriate therapeutic intervention will require genotyping many polymorphic sites in large numbers of genes or single nucleotide polymorphism sites throughout the genome. Additionally, each polymorphism will have to be associated with a phenotype. Presumably, a composite phenotype may be predicted by integrating anticipated contributions from each polymorphism contributing to the complex genotype. Methods for executing such large-scale genotyping studies are rapidly evolving and becoming available. DNA microarray technology applied in hybridization-based genotyping assays is particularly well suited to respond to the accelerating pace of polymorphism discovery and the associated demand for highly parallel genotyping capability.

Base Sequence↗
PubMed1991

Diagnostic applications of DNA probes.

This review has described several of the most common molecular biologic techniques that are, or will be, employed in the diagnostic laboratory. The potential advantages of these DNA probe assays in the diagnosis of infectious diseases include: rapid detection and identification of infectious agents; the ability to screen selected specimens using batteries of probes; and the detection of nonviable or difficult-to-culture organisms. The potential disadvantages of DNA probe assays include: the use of isotopic detection methods for optimum sensitivity; limited diagnostic sensitivity of current assays; slow turna-round time for some assay formats; expense of current reagents; limited availability of many probes; lack of technical expertise in most diagnostic laboratories; and the requirement for antimicrobial susceptibility testing (requires culture). Given the above advantages and disadvantages, there are several key issues that must be considered before adopting DNA probe technology in the diagnostic laboratory; the cost of performing routine culture and identification versus the cost of screening with probes--both the number and type of specimens and the time savings that may be realized by eliminating routine cultures; the prevalence of the infectious agent--even the best DNA probe assay may not be useful or practical in a low-prevalence situation; the need for additional equipment and space; and the interpretation of false-positive and false-negative results--additional research is needed in this area. However, laboratories must consider these issues when using a test other than the current gold standard (i.e., culture). DNA probe technology is with us and expanding rapidly. The intelligent application of this new technology will require communication between laboratorians and clinicians and careful consideration of the many advantages and disadvantages discussed above.

Blotting, Southern↗
PubMed2000

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Affinity Labels↗
PubMed2006

Protein binding microarrays (PBMs) for rapid, high-throughput characterization of the sequence specificities of DNA binding proteins.

DNA binding proteins play a number of key roles in cells, in processes including transcriptional regulation, recombination, genome rearrangements, and DNA replication, repair, and modification. Of particular interest are the interactions between transcription factors and their DNA binding sites, as they are an integral part of the transcriptional regulatory networks that control gene expression. Despite their importance, the DNA binding specificities of most DNA binding proteins remain unknown, as earlier technologies aimed at characterizing DNA-protein interactions have been time consuming and not highly scalable. We have developed a new DNA microarray-based technology, termed 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. The resulting DNA binding site data can be used in a number of ways, including for the prediction of the genes regulated by a given transcription factor, annotation of transcription factor function, and functional annotation of the predicted target genes.

Base Sequence↗
PubMed2001

Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins.

The number of proteins produced by recombinant DNA technology continues to grow at a rapid pace. In this review, the emphasis is on proteins that are of therapeutic interest. Aspects of protein analysis, such as glycoform separation of proteins produced in mammalian cells and the separation of oligosaccharides for structure elucidation, are covered. The use of antibodies as therapeutic proteins is growing and currently antibodies are the largest class of proteins produced by biotechnology. This has merited a separate section on analysis of antibodies by capillary electrophoresis (CE). Applications of mass spectrometry as an ancillary technique, used in conjunction with CE, are also covered briefly. This review covers the literature since 1999.

Biotechnology↗
PubMed2001

Real time detection of the tri5 gene in Fusarium species by lightcycler-PCR using SYBR Green I for continuous fluorescence monitoring.

LightCycler technology combines rapid in vitro amplification of DNA with real time detection and quantification of the amount of target molecules present in a sample. The system enables a 35-cycle PCR with 32 samples do be completed in 45 min, including quantification and identification of the product. It is therefore well suited for routine analysis of large numbers of samples in quality control and for defining HACCP concepts. Based on PCR primers specific to the tri5 gene, a quantitative group specific assay was established for Fusarium species producing trichothecenes. In the assay, SYBR Green I was used as fluorescent dye enabling real time detection of PCR products. Characterisation of the amplicons was achieved by melting point analysis (85 +/- 0.1 degrees C). Nonspecific products such as primer dimers could readily be distinguished from the product by their lower melting points. Composition of the amplification buffer was optimised and various hot start methods were tested in order to achieve the highest sensitivity of the assay. Uracil DNA glycosylase was added to prevent amplification of nonspecific products due to DNA carryover. The spectrum of species detected was generally in accordance with the results found in conventional PCR using the Tox5 primer pair. Reproducibility in six parallel experiments of the assay was determined to be 98% in the range between 0.05 and 6 ng of purified Fusarium graminearum DNA. The assay was used to analyse 30 wheat samples contaminated with toxigenic Fusarium spp. Contamination ranged from 0% to 78% as revealed by mycological analysis, and this is compared with results from the LightCycler. This is the first report on the use of the LightCycler system in combination with SYBR Green I for the quantification and identification of fungal DNA in pure cultures and sample material.

Fluorescence↗
PubMed2001

CYP2D6 genotyping with oligonucleotide microarrays and nortriptyline concentrations in geriatric depression.

Recent advances in oligonucleotide microarray technology ("gene chips") permit rapid screening for DNA sequence variation. The CYP2D6 gene encodes debrisoquine hydroxylase, which metabolizes the antidepressant nortriptyline and other psychotropic medications. Nortriptyline plasma concentrations were obtained after at least three weeks of treatment in 36 geriatric patients with major depression who were taking a mean of 8.6 other medications besides nortriptyline. Oligonucleotide microarrays were used to detect 16 CYP2D6 alleles that affect debrisoquine hydroxylase activity. Subjects carrying alleles encoding impaired debrisoquine hydroxylase activity had significantly greater nortriptyline concentrations and lower nortriptyline doses than did other subjects. Significant correlations were found between the numbers of alleles encoding decreased metabolism and nortriptyline plasma concentration, nortriptyline dose, and nortriptyline plasma concentration standardized for dose, indicating a gene dosage effect. These results demonstrate that CYP2D6 genotyping on a microarray platform can be used to predict plasma antidepressant concentrations despite advanced patient age and numerous concurrent medications.

Aged↗
PubMed2025

Advances in large-scale DNA engineering with the CRISPR system.

In recent years, DNA engineering technology has undergone significant advancements, with clustered regularly interspaced short palindromic repeats (CRISPR)-based target-specific DNA insertion emerging as one of the most rapidly expanding and widely studied approaches. Traditional DNA insertion technologies employing recombinases typically involve introducing foreign DNA into genes in vivo by either pre-engineering recognition sequences specific to the recombinase or through genetic crossing to incorporate the requisite recognition sequence into the target gene. However, CRISPR-based gene insertion technologies have advanced to streamline this engineering process by combining the CRISPR-Cas module with recombinase enzymes. This process enables accurate and efficient one-step insertion of foreign DNA into the target gene in vivo. Here we provide an overview of the latest developments in CRISPR-based gene insertion technologies and discusses their potential future applications.

CRISPR-Cas Systems↗
PubMed2002

Progress in high throughput SNP genotyping methods.

Most current single nucleotide polymorphism (SNP) genotyping methods are still too slow and expensive for routine use in large association studies with hundreds or more SNPs in a large number of DNA samples. However, SNP genotyping technology is rapidly progressing with the emergence of novel, faster and cheaper methods as well as improvements in the existing methods. In this review, we focus on technologies aimed at high throughput uses, and discuss the technical advances made in this field in the last few years. The rapid progress in technology, in combination with the discovery of millions of SNPs and the development of the human haplotype map, may enable whole genome association studies to be initiated in the near future.

Animals↗
PubMed2004

Applications and challenges of DNA microarray technology in military medical research.

This review discusses the challenges and applications of DNA microarray technology as it is being used in each of the four major research areas of the U.S. Army Medical Research and Materiel Command: military infectious diseases, combat casualty care, military operational medicine, and medical chemical and biological defense. The overall objective of this review is two-fold. First, the objective is to increase awareness in senior military leadership of the challenges and opportunities presented by DNA microarray technology and the emerging and rapidly changing field of bioinformatics. Second, the aim is to publicize to the civilian research community the additional challenges associated with the use of microarray technology in military medical research. This discussion contains material that would be useful for making programmatic recommendations that team strategic research investments and emerging technologies with U.S. Army Medical Research and Materiel Command resources.

Biological Warfare↗
PubMed2000

Multiple analysis of three common genetic alterations associated with thrombophilia.

We have previously reported the use of a novel mini-sequencing protocol for detection of the factor V Leiden variant, the first nucleotide change (FNC) technology. This technology is based on a single nucleotide extension of a primer, which is hybridized immediately adjacent to the site of mutation. The extended nucleotide that carries a reporter molecule (fluorescein) has the power to discriminate the genotype at the site of mutation. More recently, the prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia. This study describes the use of the FNC technology in a combined assay to detect factor V, prothrombin and MTHFR variants in a population of Australian blood donors, and describes the objective numerical methodology used to determine genotype cut-off values for each genetic variation. Using FNC to test 500 normal blood donors, the incidence of Factor V Leiden was 3.6% (all heterozygous), that of prothrombin 20210 was 2.8% (all heterozygous) and that of MTHFR was 10% (homozygous). The combined FNC technology offers a simple, rapid, automatable DNA-based test for the detection of these three important mutations that are associated with familial thrombophilia.

Adenine↗
PubMed2003

[Use of molecular biological methods in invasive prenatal genetic diagnosis].

In the past, prenatal cytogenetic analysis was limited by answering times of one to three weeks and lack of exact diagnosis of some structural abnormalities. The number of prenatal analyses of monogenic diseases was small due to lack of knowledge of the gene and the mutation in question. The introduction of molecular biological techniques allows prenatal diagnosis of the most frequent trisomies within one to two days and exact diagnosis of almost all structural abnormalities. The Human Genome Project now allows prenatal diagnosis for most monogenic diseases, and the rapid improvement of the DNA-chip technology will increase the number of prenatal diagnoses even further.

Chromosome Aberrations↗
PubMedDate not supplied

Nanotechnologies and microchips in genetic diseases.

Microarrays or microchips represent a new area of high technology, which will completely change the methodological approach to basic research and clinical diagnostics. This technology can be used for genotyping, expression profiling and proteome analysis. Genetics and molecular medicine have an expanding need for rapid genotyping, mutational analysis and DNA re-sequencing technologies, i.e. microarrays that have a clear potential for miniaturization, parallelization, and automation and enable high-throughput screening. Expression profiling technology is a new tool for investigating expression patterns, identifying new disease genes either for monogenic disorders or for complex traits, identifying new functional and cellular relationships and identifying new pathways and possible related drugs. This technology has been successfully applied to the study of complex traits, i.e. cardiovascular diseases, cancer and type II diabetes, providing new insights into possible pathogenetic mechanisms and new therapeutical approaches. Finally, microarray can further improve proteome analysis. This review discusses these points.

Gene Expression Profiling↗
PubMed2004

[Future perspective on the prevention of nephrolithiasis].

Renal stone formation has been explained by the physicochemical theory; i.e., nucleation, growth and aggregation of crystals in the urine. Current medical prevention is based on this theory and seeks to modulate promoters and inhibitors of stone formation. Recent studies have identified increasing numbers of macromolecular inhibitors such as glycosaminoglycans, bikunin, osteopontin and urinary prothrombin F1. These appear to be more important than low-molecular inhibitors like citrate. On the other hand, many investigators have focused on the role of tubular epithelial cells in stone formation. While crystal retention in the nephron has been considered necessary for stone formation, we and others have found that calcium oxalate crystals can bind to renal epithelial cells. Moreover, available evidence suggests that oxalate and/or calcium oxalate crystals can damage renal epithelial cells and enhance crystal binding. Concurrently, oxalate exposure induces genes coding macromolecular inhibitors, which are supposed to be a protective mechanism against stone formation. Thus, understanding of the mechanisms involved in kidney stone formation could lead to new therapeutic approaches to preventing stone recurrence. However, clinical application of the prophylactic approaches proposed here awaits further progress in basic research using rapidly growing new technologies. Application of the DNA microarray technique to the rat stone model may provide a clue to understanding stone forming process at the genetic level. In addition, case-control association analysis using single nucleotide polymorphism as a marker may be useful for detecting susceptibility genes in patients with calcium stone disease.

Allopurinol↗
PubMedDate not supplied

Symposium on microbiology update: old friends and new enemies. Listeria monocytogenes.

Listeria monocytogenes, one of the "new enemies" in food microbiology, is a human and animal pathogen that is widespread in nature. The organism is a transient constituent of the intestinal flora excreted by 1-10% of healthy humans. It is an extremely hardy organism and can survive for many years in the cold in naturally infected sources. L. monocytogenes has been isolated from a wide variety of foods, including dairy products, meats, and fish. Although most of the foodborne listeriosis outbreaks have been linked to the consumption of dairy products, recent sporadic cases have been associated with meats, as well as other foods. It is now recognized that listeriolysin 0, a 60-kilodalton protein, is one of the major virulence factors of the organism. All strains of L. monocytogenes are pathogenic by definition although some appear to be more virulent than others. There have been recent reports of hemolytic isolates of L. monocytogenes, which are nonpathogenic for mice. Attachment to and penetration of cells also appear to be prerequisites for human infection. Cultural methodology for isolating the organism from foods has been in a state of flux since 1985. Rapid methods using both ELISA and DNA technology have been developed. Because of the widespread nature of the organism, it is nearly impossible to eliminate it from the food supply. However, by using a hazard analysis-critical control point approach, the health hazard associated with this organism can be reduced to a minimum.

Food Microbiology↗
PubMed1987

The molecular biology of the colony-stimulating factors.

Our understanding of the biochemistry and molecular biology of the colony stimulating factors (CSF) and the regulation of their production has advanced rapidly with the application of recombinant DNA technology. This report reviews several aspects of the regulation of CSF production and the structural features relating to their function. The structural gene for hGM-CSF is present as a single copy, is 2.5 kilobase long region and is located on the long arm of chromosome 5. The genes for human multi-CSF and M-CSF are also on chromosome 5. The gene for hGM-CSF is organized into four exons. The intron-exon boundaries contain a single consensus splice donor and acceptor sequence. CSFs are elaborated by endothelial cells, fibroblasts, and T lymphocytes in response to inflammatory mediators. Many of these same cell types respond to inflammatory stimuli with production of additional immune mediators such as IL-1, IL-2, or the interferons. Insights are now emerging into important structure--function relationships of the hematopoietic growth factors and molecular events in the regulation of CSF gene expression.

Chromosomes, Human, Pair 5↗