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PubMed2004

Surface-attached molecular beacons light the way for DNA sequencing.

Rapid testing of DNA and RNA nucleotide sequences is required for various research protocols including wide-scale genetic testing, diagnostics, fast detection of biological warfare agents, environmental testing and forensic medicine. At present many laboratories are interested in research and development of an inexpensive, easy-to-use, fast-response device for this purpose. Various methods based on acoustic, electronic and optical detection of the DNA hybridization event have been reported.

Acoustics↗

PubMed1977

Is evolution gradual or rectangular? Evidence from living fishes.

The traditional view that most evolutionary change is gradual and cumulative within lineages (phyletic gradualism) has recently been challenged by the proposition that the majority of evolutionary change is concentrated within speciation events (rectangular evolution). The logical implications of these competing hypotheses for the means and variances of genetic distance among living members of rapidly and slowly speciating phylads are examined. An example of a critical test of gradual versus rectangular evolution is provided by electrophoretic analyses of genic composition in 69 species of North American Cyprinidae (minnows), and 19 species of Centrarchidae (sunfish). Rate of protein evolution appears somewhat decelerated, if anything, in the rapidly speciating minnows. Results are inconsistent with predictions of rectangular evolution, but are not demonstrably incongruent with predictions of phyletic gradualism.

Animals↗

PubMed2005

Inherited risk factors for venous thrombosis.

Venous thrombosis occurs as a consequence of genetic and environmental risk factors. Since the discovery of factor V Leiden, the most common genetic risk factor, there has been intense interest in clarifying the roles of genes and the environment with thrombosis risk. The translation of this risk information to clinical practice is a challenging one in the setting of a rapidly expanding knowledge base that includes application of genetic medicine. There are benefits, but also potential harms, of testing for inherited disorders associated with thrombosis. This paper reviews inherited risk factors for thrombosis and discuss clinical applications of testing.

Environment↗

PubMed1993

Fetal cells in the maternal circulation. Technical considerations for practical application to prenatal diagnosis.

Recent advances in cell separation technology and DNA analytic techniques leave little doubt as to the presence of fetal cells in the maternal circulation. The potential of using these cells for genetic analysis is compelling. The practical aspects of establishing a universal method utilizing the new capabilities in clinical practice have not been addressed to date. The major hurdles that still need to be traversed before this technology is universally adopted include the identification of appropriate sampling and separation methods yielding fetal cells amenable to genetic analysis by rapid DNA technologies, clinical studies of appropriate statistical power to validate and compare this approach to current genetic testing, and comparison of this approach to other noninvasive paradigms such as triple screening. Despite the tremendous value of noninvasive genetic screening, the rigorous course required to progress from description of scientific capability to validation of a clinical test must not be ignored or rushed for financial considerations.

DNA↗

PubMed2003

Oral manifestations of genodermatoses.

Many genodermatoses have distinct oral features that may help identify or confirm a genetic diagnosis. Oral features of the disorders described here are summarized in Table 1. These conditions provide clear examples of rapid progress in the field of genetic technology relevant to patient care. Less than a decade ago, the exact genetic locus of most of these disorders was unknown. Today, for many of these disorders, the exact location of the disease-causing mutation is known and clinical genetic testing is available for patients. This information has impact not only for genetic counseling and anticipatory medical care, but also provides insight into the mechanisms of disease. How this rapid progress will impact care, and ultimately treatment of patients, remains to be seen.

Ectodermal Dysplasia↗

PubMed2002

Rapid spread of immigrant genomes into inbred populations.

When local populations are genetically differentiated from one another and partially inbred, as typically occurs in subdivided populations, immigrant genomes are predicted to be at a frequency-dependent fitness advantage due to heterosis (hybrid vigour) in their descendants. We tested this prediction with pedigreed laboratory populations of the butterfly Bicyclus anynana and report here on a rapid increase over five generations in the contribution of an initially rare immigrant genome to the local population gene pool. The replicated experimental design, including immigrant controls, demonstrates that the mechanism underlying immigrant genome spread is heterosis, and that the advantage to the immigrant genes is sustained over several generations. Our result suggests that effective migration rates may often be much higher than the numbers of individual migrants assumed by classical population genetics models, with implications for the persistence and evolution of metapopulations.

Animals↗

PubMed2006

Characterization of LF9, an octoploid strawberry genotype selected for rapid regeneration and transformation.

Cultivated strawberry (Fragaria xananassa) is a valuable crop, yet the absence of a rapid, high-throughput transgenic system has precluded meaningful application of biotechnology and translation of information from plant models to this crop. A new octoploid strawberry genetic line Laboratory Festival #9 has been identified, selected solely for its rapid regeneration and efficient transformation. Direct organogenesis has been achieved from all tissues tested, with rapidly-growing shoot initials visible in as few as 13 days. The conditions for optimal shoot regeneration, transformant selection, root generation, and plant acclimatization are presented. The progression from explant to plant in soil can be achieved in about 60 days. The development of transformation protocols in this rapid-cycling genotype allows high-throughput studies of gene function in the octoploid strawberry genetic background.

Acclimatization↗

PubMed1999

Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry.

The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them a valuable source of genetic markers for identity testing, genome mapping, and medical diagnostics. Conventional technologies for detecting SNPs are laborious and time-consuming, often prohibiting large-scale analysis. A rapid, accurate, and cost-effective method is needed to meet the demands of a high-throughput DNA assay. We demonstrate here that analysis of these genetic markers can now be performed routinely in a rapid, automated, and high-throughput fashion using time-of-flight mass spectrometry and a primer extension assay with a novel cleavable primer. SNP genotyping by mass spectrometry involves detection of single-base extension products of a primer immediately adjacent to the SNP site. Measurement of the mass difference between the SNP primer and the extension peak reveals which nucleotide is present at the polymorphic site. The primer is designed such that its extension products can be purified and chemically released from the primer in an automated format. The reduction in size of the products as a result of this chemical cleavage allows more accurate identification of the polymorphic base, especially in samples from a heterozygotic population. All six possible heterozygotes are resolved unambiguously, including an A/T heterozygote with extension products differing by only 9 Da. Multiplex SNP determination is demonstrated by simultaneously probing multiple SNP sites from a single polymerase chain reaction (PCR) product as well as from multiplexed PCR amplicons. Samples are processed in parallel on a robotic workstation, and analyzed serially in an automated mass spectrometer with analysis times of only a few seconds per sample, making it possible to process thousands of samples per day.

DNA Primers↗

PubMed1996

Generation and mapping of Mus spretus strain-specific markers for rapid genomic scanning.

We describe here a set of genetic markers, based on IRS-PCR amplification difference, that are specifically designed for efficient, high throughput genetic mapping in [(M. domesticus x wild-derived) F1 x M. domesticus] interspecific backcrosses. 146 new genetic loci have been mapped, and strain distribution for these markers has been determined in 96 mouse strains. 103 (81%) of 127 tested markers are present only in one or more wild-derived strains, but absent in 76 other commonly used strains, demonstrating their utility in a variety of mouse pair combinations. Because of the ease of genotyping with this marker set, rapid genome scans for complex genetic trait loci involving crosses between wild-derived strains and other commonly used strains can now be carried out efficiently with large numbers of animals.

Animals↗

PubMed1999

Rapid genetic screening for hemochromatosis using automated SSCP-based capillary electrophoresis (SSCP-CE).

Hereditary hemochromatosis (HHC) represents an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. The recently detected association between two point mutations in the HFE gene on chromosome 6p and HHC has made it possible to screen for the disease before the onset of irreversible tissue damage. Conventional genetic testing is based on restriction fragment-length polymorphisms (RFLP) using two endonuclease recognition sites in codon 63 or 282, respectively. In this study, we have adapted single-strand conformation polymorphism analysis for capillary electrophoresis (SSCP-CE) to detect homozygote or heterozygote point mutations. Two HFE gene fragments spanning codons 63 and 282 were amplified by a duplex PCR using genomic DNA from peripheral blood or from tissue sections of paraffin-embedded liver biopsies as template. Thereby, rapid genotyping of both HFE mutations was achieved with a single PCR, omitting the need of further analysis by restriction digest. Eighty-five patients with liver disease and/or suspected iron overload were genotyped using SSCP-CE, and all results were verified by conventional RFLP analysis. In summary, SSCP-CE proved to be a reliable, cost-effective, sensitive and rapid method for genotyping HFE mutations. This method will further facilitate high-throughput genetic screening using capillary array electrophoretic devices.

Adult↗

PubMed2000

Obstetrician-gynecologists' opinions and attitudes on the role of genetics in women's health.

Our objective was to describe gynecologists' current practice patterns and opinions on genetic screening and their perceived importance of genetic screening within individual practices. A questionnaire survey was sent to 1248 American College of Obstetrics and Gynecology (ACOG) Fellows, of whom 564 (45%) responded. Results from the 428 respondents providing genetic screening for heritable diseases or disorders are reported. Forty-four percent of respondents believe advances in the treatment of genetic diseases are likely in the next 10 years. Currently, however, genetics in gynecological practice receives infrequent attention. Twenty-four percent of respondents do not routinely review family histories at gynecological visits, 39% rate genetic issues as last among priorities in the office, and only 14% obtain consent for the DNA tests that they initiate. Although 21.3% identified themselves as sole providers of genetic information and counseling to their patients, most (65.4%) note they are not confident of their knowledge of genetics, particularly concerning breast and ovarian cancer. For obstetrician-gynecologists to keep pace with the rapid changes in genetics, further education and assimilation of genetics into the routine office practice will need to occur. Not currently viewed as a priority among practitioners, issues of genetic knowledge, ethics, and test interpretation will soon need attention. National organizations, continuing medical education, and existing genetic centers will need to meet these recognized demands.

Adult↗

PubMed2004

Strategies for the rapid prenatal diagnosis of chromosome aneuploidy.

Rapid diagnosis of common chromosome aneuploidies in raised risk pregnancies, usually prior to full karyotype analysis, is now carried out in a number of European genetic centres; several techniques for detecting genomic copy number changes have been described. Prenatal diagnosis of genetic disease requires accurate and robust assays; the invasive procedures are associated with a risk of pregnancy loss and an abnormal result may lead to termination of the pregnancy. The testing of prenatal material (amniotic fluid, chorionic villi or, more rarely, fetal blood) is associated with specific problems, including the quality and quantity of the tissue and difficulties of interpretation due to phenomena such as maternal cell contamination and mosaicism. In addition, there are 24-h, high-throughput demands on centres offering such a service. The extent to which existing and proposed strategies, including different PCR-based assays, a multiplex ligation-dependent probe amplification approach, and microarrays, fulfil the requirements of rapid prenatal testing is discussed. In the past 3 years, we have tested 7720 prenatal samples for trisomies 13, 18 and 21 using a quantitative fluorescence-PCR (QF-PCR) approach. The abnormality rate was 5.7%. There were no misdiagnoses for nonmosaic trisomy, the amplification failure rate was 0.09% of samples, and 97% of samples received a report on the working day following sample receipt. Maternal cell contamination and mosaicism were also detected. Our data recommend a QF-PCR approach as the current method of choice for rapid aneuploidy testing.

Aneuploidy↗

PubMedDate not supplied

EILATox-Oregon Workshop: blind study evaluation of Vitotox test with genotoxic and cytotoxic sample library.

In order to assess the robustness, sensitivity and specificity of a recently developed Vitotox test, 17 blind coded chemicals and three environmental water samples were tested at the EILATox-Oregon Workshop using the Thermo Electron Vitotox kit. The Vitotox test is a rapid geno- and cytotoxicity test using standard 96- or 384-well microtitre plates. The genotoxicity test is based on two genetically modified Salmonella typhimurium strains containing bacterial luciferase operon from Vibrio fisheri under the SOS inducible promoter. The SOS system is an inducible network in Escherichia coli that responds to DNA damage and activates DNA repair. The Vitotox genotoxicity test bacteria strain carries bacterial luciferase genes under the control of SOS inducible promoter and therefore any DNA damage inside the cells induces the production of bacterial luciferase. The luciferase expression is then followed with a microtitre plate luminometer for 3 h after mixing different dilutions of sample with the test bacteria. The genotoxicity index is calculated for each dilution and the genotoxicity of the sample is interpreted based on kinetic time curves and genotoxicity vs concentration/dilution curves. Cytotoxicity of the sample is determined simultaneously with another test strain containing the same luciferase operon controlled by the constitutive promoter. This bacterium produces constant bioluminescence and any decrease of the bioluminescence production is used as a marker for cytotoxicity. As a miniaturized microtitre plate assay the Vitotox test requires a very small quantity of the sample material. The samples used in the workshop were diluted 1 : 10 or 1 : 100 before testing. Genotoxicity and cytotoxicity data were collected at dilutions of 1 : 10-1 : 2000. When the samples of the EILATox-Oregon Workshop were tested using the Vitotox test, four coded chemicals out of 17 were determined to be genotoxic. Seven chemicals and one environmental sample were found to be cytotoxic. Three chemical samples were found to be both geno- and cytotoxic.

Aliivibrio fischeri↗

PubMed2003

Molecular detection of resistance to antituberculous therapy.

Drug-resistant tuberculosis is becoming increasingly common and represents a worldwide threat. Therefore, new approaches for the rapid susceptibility testing of Mycobacterium tuberculosis are needed to replace traditional culture-based methods. This article presents the genetic background of drug resistance in tubercle bacillus, and the methods currently available for genotypic susceptibility testing.

Antitubercular Agents↗

PubMed2000

A genotypic analysis of patients receiving Zidovudine with either Lamivudine, Didanosine or Zalcitabine dual therapy using the LiPA point mutation assay to detect genotypic variation at codons 41, 69, 70, 74, 184 and 215.

BACKGROUND: The Murex-Innogenetics LiPA HIV-1 RT assay can be used to identify the presence of mutations of the reverse transcriptase gene at codons 41, 69, 70, 74, 184 and 215 of HIV-1, which have been shown to confer resistance to the nucleoside analogs Zidovudine (ZDV), Lamivudine (3TC), Didanosine (ddI) and Zalcitabine (ddC). The M184V mutation of the reverse transcriptase gene of HIV-1 has been associated with resistance to 3TC, ddC and ddI. This mutation has also been observed in patients receiving ZDV+ddC and ZDV+ddI. We used LiPA HIV-1 RT assay to identify the presence of either consensus methionine 184 or the mutant valine 184 with three groups of patients who were treated with ZDV/3TC, ZDV/ddI or ZDV/ddC combination therapy. OBJECTIVES: The aim of our study was to determine the viral genotype of patients who were considered to be failing therapy, by two ways: using sequencing and LiPA assays. In particular we were interested in establishing a possible correlation between these methods. STUDY DESIGN: The study group consisted of a consecutive series of 33 patients with a treatment failure, 18 of whom received ZDV+3TC therapy, seven received ZDV+ddI and eight received ZDV+ddC therapy. We also examined a small cohort of seven seroconverters. RESULTS: The M184V mutation was observed in 47.0% of patients receiving ZDV+3TC combination therapy but was not observed in either patient group receiving either ddI or ddC as co-therapy with ZDV. There was no evidence of the L74V mutation in our study group in either the ZDV/ddI or ZDV/ddC combination therapy group. We found the frequency of the K70R mutation to be higher in patients treated with ZDV/ddI (P=0.033) or ZDV/ddC (P=0.3) when compared with patients treated with ZDV/3TC. CONCLUSION: The LiPA assay allowed for the rapid detection of wild type and amino acid variations at key positions conferring resistance to the most used antiviral RT inhibitors. This represented a rapid, quite sensitive, and simple genotyping test. For these reasons the LiPA assay proved to be useful in studying genetic resistance in large screenings, when key RT mutations could be useful in guiding an effective HIV-1 suppressing regimen.

Anti-HIV Agents↗

PubMed2002

Evidence of en bloc duplication in vertebrate genomes.

It has been 30 years since it was first proposed that the vertebrate genome evolved through several rounds of genome-wide duplications (polyploidizations). Despite rapid advances in genetics, including sequencing of the complete genomes of several divergent species, this hypothesis has not been tested rigorously and is still a matter of debate. If polyploidizations occurred during chordate evolution, there should be a network of paralogous regions in the present-day jawed vertebrate (Gnathostomata) genomes. Here we present an investigation of the major histocompatibility complex (MHC) paralogous regions, which we accomplished by characterizing the corresponding region in amphioxus by identifying nine anchor genes and sequencing both the anchor genes and the regions that flank them (a total of 400 kb). Phylogenetic analysis of 31 genes (including the anchor genes) in these regions shows that duplications occurred after the divergence of cephalochordates and vertebrates but before the Gnathostomata radiation. The distribution of human and amphioxus orthologs in their respective genomes and the relationship between these distributions support the en bloc duplication events. Our analysis represents the first step towards demonstrating that the human ancestral genome has undergone polyploidization. Moreover, reconstruction of the pre-duplicated region indicates that one of the duplicated regions retains the ancestral organization.

Animals↗

PubMed1991

[STAPHYOGRAM, a new rapid identification kit for the aerobic, gram-positive, catalase-positive cocci--application of fluorometric microplate hybridization for the pre-identification of 386 isolates used].

A new simplified test kit, STAPHYOGRAM plate, was developed for 4-hr identification of aerobic, Gram-positive and catalase-positive cocci. The plate has 18 wells, in which different dehydrated substrates and nutrients are fixed. An 18-hr agar-culture suspension of a test strain with a turbidity of McFarland No. 4 was distributed into all wells in 50-microliters quantities. After 4-hr incubation at 37C, the profile number was obtained by summarizing positive reactions. The ability of the plate to differentiate the type strains of the 30 species of the three genera in the family Micrococcaceae was confirmed. These three genera are Staphylococcus, Micrococcus and Stomatococcus. The applicability of the fluorometric microplate hybridization technique to identification of aerobic, Gram-positive and catalase-positive cocci was confirmed by homologous hybridization among the type strains of the 30 species. Thus, 386 isolates of human and animal origin were pre-identified by microplate hybridization and used for evaluating the STAPHYOGRAM plate. Of the 236 profile numbers thus obtained with the 386 isolates, 218 (92.4%) were species-proper each and all for the 15 species of Staphylococcus and Stomatococcus mucilaginosus. A total of 342 (88.6%) of the 386 isolates were given such profile numbers, and were identified without any additional test. Among the 15 species identified primarily by the results of STAPHYOGRAM plate culture, S. caprae, S. lugdunensis, S. gallinarum and S. delphini were validly published after Approved Lists of Bacterial Names. The identified strains of S. caprae (48), S. haemolyticus (46), S. capitis (35) numbered between those of S. epidermidis (67) and S. saprophyticus (31). Profile numbers common to two species were seven (27 strains) and that to four species was one (17 strains). These 44 strains were identified with one to three additional tests. From these results, we were convinced that the STAPHYOGRAM test plate is useful for the rapid identification of members of family Micrococcaceae. By compiling STAPHYOGRAM plate data on genetically identified strains, an exclusive list of profile numbers will soon be prepared for perfection of the kit.

Animals↗

PubMed2001

Miniaturization technologies for molecular diagnostics.

BACKGROUND: Molecular diagnostics devices are becoming smaller. With the advancement of miniaturization technologies, microchip-based systems will soon be available for genetic testing. The purpose of this review is to highlight the underlying principles in miniaturization, the strategies being developed for bioanalysis, and the potential impact on the practice of this rapidly growing medical discipline. APPROACH: The author discusses DNA microchips and their practical importation into the clinical laboratory, based on his background in medical device and microchip design and development. His discussion is supported by a body of literature covering both biomedical and electrical engineering and more recent publications in the field of molecular genetics and pathology. CONTENT: This review is descriptive and intended to outline the technologic and methodologic approaches to the creation of an integrated genetic analysis instrument based on miniature components. The review draws on published scientific evaluations of these devices without regard to the companies involved in their development. SUMMARY: The intent of this review is that the reader will better understand the variety of technical approaches toward the miniaturization of molecular genetic testing for the clinical laboratory. With insight into the principles underlying the operation of these chips and the integrated systems, the end user can better evaluate the value to the field in terms of making molecular genetics testing simpler, faster, and less expensive.

Biosensing Techniques↗