Detection Of Specific Genes Research Articles

Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction

Use of the polymerase chain reaction, coupled with flow cytometry, to detect a plasmid encoded xylE gene sequence in intact cells of Escherichia coli and Pseudomonas putida was investigated. Optimal incorporation of fluorescently labelled dUTP into a full length PCR product required substitution at a level of 2:3 dUTP:dTTP. Formaldehyde fixed cells of both species were counted before and after thermal cycling. Sufficient numbers of cells remained intact for subsequent detection using microscopy and flow cytometry but light scatter properties were altered. Intact cell suspensions of both species containing plasmid pLV1013 were subjected to thermal cycling with fluorescent dUTP in the reaction mix. Subsequent analysis by flow cytometry allowed detection of a fluorescent PCR product associated with cells. Control samples (without the plasmid) showed only background fluorescence. This method demonstrates the potential for applying DNA amplification methods for sensitive detection of specific sequences localized inside intact bacterial cells.

Chemical Ecology: Possible Linkage between Macro- and Microbial Ecology

Microbes are often the largest biomass in an ecosystem, yet are not often considered in classical ecology. Problems with the study of the ecology of microbes come from the difficulties in applying the isolation and culture techniques that were so successful in medical studies but are not effective in the environment. Most environmental microbes simply will not grow in culture. The problem of assessing the impact of microbes on ecosystems is further complicated as the most significant impact of a microbial community on an ecosystem is the result of metabolic activities. Inactive microbes can be a food source. but the biogeochemical metabolism of the active microbes have the greatest impact on the environment. In stable soils and sediments, only a tiny proportion of the potential metabolic capacity and versatility of that community is being actively expressed at a given time. Microbes in these environments are poised to rapidly take advantage of nutrients that become available following a disturbance. This dramatic reaction to disturbance makes measuring the metabolic activity of microbes in undisturbed soil a significant challenge. Heterogeneity on the scale of the microbes in the distribution of nutrients, carbon sources, water, ions, or terminal electron acceptors in soils is a significant complication. The assumptions of colligative properties of solutions for soil environments in the estimations of kinetics is one of the reasons that engineers have so much trouble making successful models for in situ bioremediation in these heterogeneous soils and sediments. How then, can soil microbial community ecology be approached ? The hypothesis that many of the responses of monocultures of microbes to specific conditions will predict the responses of these organisms to specific conditions in the soil community seems to hold true. This theory is being tested using genetically engineered microbes with reporters in biofilms and can be examined in soils, if the analysis does not apply selective pressures that can distort the in situ conditions. The knowledge of physiological responses of monocultures, and the structural modifications they produce, provides a mechanism to predict the responses of a community to specific change. In characterizing the microbial community in situ, there are advantages to analyzing lipid membranes, because all viable cells are enclosed in lipid membranes containing polar lipids. If specific organisms have sufficiently unusual lipids in their membranes then these lipids can serve as signatures to define the community structure. If there are specific conditions that result in structural modifications of the lipids, then the detection of these modifications can be utilized to define the ecology of the microniche of that organism and thus of at least a portion of the microbial community. This gives insight into the microbes' nutritional/physiological status and the suitability of the local environment. The presence of polar lipids can define the viable biomass, as no cell can function without an intact membrane which contains polar lipids. The activation of endogenous phospholipase activity with cell death can leave traces of the recent lysis in the diglyceride structure. The detection of specific genes can define the limits of adaptation but not always reflect current metabolic activity

Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.

DNA sensor: A novel electrochemical gene detection method using carbon electrode immobilized DNA probes

Abstract The authors have developed a novel, rapid, convenient, and specific gene detection method, named the ‘DNA sensor,’ using a graphite electrode loaded with DNA probes. Synthesized oligonucleotide (5-TGCAGTTCCGGTGGCTGATC-3′) complementary to oncogene v-myc was employed for a model probe. The oligonucleotide was chemically adsorbed on a basal plane pyrolytic graphite (BPPG) electrode. The sensor was able to be applied to a hybridization reaction (40°C) in a linearized pVM623 solution carrying the Pst I fragment of v-myc (1.5 kbp). After the hybridization reaction, the sensor was immersed into an acridine orange solution (1 μM) and washed with a phosphate buffer (pH 7.0). Acridine orange intercalated between base pairs of the formed double stranded DNAs on the electrode. The anodic peak potential of acridine orange that interacted with the DNAs on the electrode was measured. The positive shift of the peak potential increased in proportional to the pVM623 concentration in the hybridization reaction. 10...

Automation of specific human gene detection

An instrument/chemistry system is described that automates a new chemical procedure functionally equivalent to Southern blotting. A fluorescence gel scanner that detects migrating DNA fragments in real-time analyzes the samples produced by a prototype liquid-handling instrument that automates a solution-phase hybridization/solid-phase capture chemistry for DNA analysis. The combination of this chemistry, the gel scanner, and robotic automation eliminates the tedium encountered in traditional manual methods for specific gene detection and reduces analysis time from days to hours. Restriction fragment lengths are measured with high precision by comparison with in-lane standards to minimize effects attributable to migration anomalies. The utility of this automated system is demonstrated by executing a clinical research application involving hybridization to a multi-copy repeat sequence on the Y chromosome and its detection.