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Developing nucleic acid-based electrical detection systems
Get PDFDevelopment of nucleic acid-based detection systems is the main focus of many research groups and high technology companies. The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices. From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive. New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit. Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field. Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology. The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection. Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health. Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector. In this review some developments and current directions in nucleic acid-based electrical detection are discussed
The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p.
Get PDFThe mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast
Gene-based identifcation of bacterial colonies with an electric chip
Get PDFA method for the identiWcation of bacterial colonies based on their content of speciWc genes is presented. This method does not
depend on DNA separation or DNA ampliWcation. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin
was identiWed with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads,
followed by enzymatic labeling and measurement of theenzyme product with a silicon-based chip. An hblC-positive colony containing
107 cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is
illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize
the DNA fragmentation as a function of ultrasonication time. The Wrst minutes of sonication increased the signal due to both
increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diVusion and
faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on
the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system
integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality
Atypical microbial infections of digestive tract may contribute to diarrhea in mucopolysaccharidosis patients: a MPS I case study.
Get PDFBACKGROUND:Mucopolysaccharidoses are heritable, metabolic diseases caused by deficiency in an activity of one of specific lysosomal enzymes involved in degradation of mucoplysaccharides (glycosaminoglycans). Among many medical problems of patients with mucopolysaccharidoses, there are frequent episodes of diarrhea of unknown etiology.
CASE PRESENTATION:A girl, diagnosed enzymatically for mucopolysaccharidosis type I (deficiency of alpha-L-iduronidase) at the age of 3 years and 9 months, was investigated until the age of 5 years and 4 months. Frequent loose stools and episodes of diarrhea, often accompanied by vomiting, were encountered. Detailed microbiological analyses were performed and atypical microbial infections (most often enetropathogenic Escherichia coli, but also other species, like Pseudomonas aeruginosa or Staphylococcus aureus, as well as adenoviruses) of the digestive tract were found in most severe diarrhea episodes. Often, isolations of pathogenic bacterial strains from stools of the investigated patient suffering from diarrhea were not obvious during the first screening, and only detailed microbiological studies, including re-isolation of colonies, gave the results of isolation of particular pathogenic strains (especially in the case of enetropathogenic E. coli).
CONCLUSION:We conclude that atypical microbial infections of digestive tract may contribute significantly to diarrhea in mucopolysaccaridosis patients. Since isolated strains were not typical and their isolation was often possible only after detailed investigation (not during a standard screening), such atypical microbial infections of digestive tract of mucopolysaccharidosis patients could be usually overlooked to date. Importantly, these atypical infections could be effectively treated with antimicrobial agents
Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips
Get PDFInfections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4 h assay time, a useful level of sensitivity (107–108 phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay
Functional and physical interactions of Faf1p, a Saccharomyces cerevisiae nucleolar protein.
No full textWe report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae. We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait. The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA. The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites
Electric chips for rapid detection and quantification of nucleic acids
Get PDFA silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the biorecognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG–alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 1011 to 1010 molecules, were assayed within 25 min and 4 h, respectively
High coding density on the largest Paramecium tetraurelia somatic chromosome.
No full textParamecium, like other ciliates, remodels its entire germline genome at each sexual generation to produce a somatic genome stripped of transposons and other multicopy elements. The germline chromosomes are fragmented by a DNA elimination process that targets heterochromatin to give a reproducible set of some 200 linear molecules 50 kb to 1 Mb in size. These chromosomes are maintained at a ploidy of 800n in the somatic macronucleus and assure all gene expression. We isolated and sequenced the largest megabase somatic chromosome in order to explore its organization and gene content. The AT-rich (72%) chromosome is compact, with very small introns (average size 25 nt), short intergenic regions (median size 202 nt), and a coding density of at least 74%, higher than that reported for budding yeast (70%) or any other free-living eukaryote. Similarity to known proteins could be detected for 57% of the 460 potential protein coding genes. Thirty-two of the proteins are shared with vertebrates but absent from yeast, consistent with the morphogenetic complexity of Paramecium, a long-standing model for differentiated functions shared with metazoans but often absent from simpler eukaryotes. Extrapolation to the whole genome suggests that Paramecium has at least 30,000 genes
Recent advances on an electric silicon based chip for analysis of nucleic acids
Get PDFRecently, several innovative designs for nucleic acid-based
electrochemical sensing have appeared. These types of sensors combine nucleic acid layers with electrochemical transducers to produce a biochip
Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization
Get PDFUnlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-aminophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step