Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Target enrichment sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources
PubMed1997

Detection of Salmonella spp. in food products by polymerase chain reaction and hybridization assay in microplate format.

Here, hybridization assay of amplified products is described which detect Salmonella spp. from chicken fillets and other food homogenates within 24 h. This technique is composed of four steps: (1) sample is pre-enriched overnight in phosphate buffered peptone water; (2) total DNA is extracted; (3) a Salmonella spp. specific DNA target sequence is amplified by polymerase chain reaction; (4) amplified products are captured by a probe covalently bound onto NH-Covalink (Nunc, Danemark) microwells and detected by a chemiluminescent enzymatic reaction. This hybridization of amplified products was demonstrated as sensitive as their analysis on agarose gel. Compared to a bacteriological method for Salmonella spp. detection, its specificity was estimated at 100% and its sensitivity was 93.2% from analysis of 207 naturally contaminated chicken fillets samples.

Animals↗
PubMed2004

Molecular approach for analysis of model fungal genes encoding ligninolytic peroxidases in wood-decaying soil systems.

AIMS: Test the use of nondegenerated consensus polymerase chain reaction (PCR) primers targeting lip and mnp sequences to detect ligninolytic fungi in wood-decaying soil systems, avoiding the need for enrichment or isolation on traditional fungal media culture. METHODS AND RESULTS: The PCR primers were tested with total DNA isolated from incubations of wood-soil systems inoculated or not with the white-rot fungi Phanerochaete chrysosporium, or a white-rot sample obtained from a Nothofagus forest. The PCR products for lip and mnp sequences were only obtained in soil with P. chrysosporium-colonized wood chips. In these soil samples, reverse transcription-PCR analysis of lip and mnp PCR products indicated expression of LipA, LipB, LipJ and MnP isoenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first assessment of the use of consensus PCR primers for direct detection of ligninolytic peroxidase genes in wood-decaying soil systems.

Basidiomycota↗
PubMed1994

Endosomal proteolysis of insulin by an acidic thiol metalloprotease unrelated to insulin degrading enzyme.

Although insulin is degraded as a consequence of receptor-mediated endocytosis, the location and nature of the responsible proteinase(s) remain controversial. Insulin degrading enzyme (IDE; EC 3.4.22.11), a mainly cytosolic neutral thiol metalloendopeptidase of 110 kDa, has been proposed to be the main cellular clearance mechanism. However, endosomes concentrate and degrade internalized insulin demonstrating that IDE is unlikely to be the relevant enzyme for endosomal proteolysis of internalized insulin in liver parenchyma. In purified endosomal fractions insulin was actively degraded at acid pH and IDE was undetectable as evaluated by immunoblotting, immunoprecipitation, or chemical cross-linking procedures. Affinity purified endosomal acidic insulinase displayed a pH optimum of 4-5.5, a lack of inhibition by EDTA and N-ethylmaleimide, and a partial metal-ion requirement (for Mn2+) all of which distinguished it from IDE. A small but detectable presence of IDE in particulate nuclear (N) and large granule (ML) fractions was observed by differential centrifugation. By analytical centrifugation, IDE cosedimented with the organelle containing the peroxisomal marker proteins catalase and thiolase (median density, 1.21 g.cm-3). By preparative centrifugation, highly purified peroxisomes were observed to be enriched in IDE. Since all cloned cDNAs of IDE (human, rat, and Drosophila) reveal a deduced classical peroxisomal targeting sequence A/SKL at their carboxyl termini this may account for the peroxisomal location of IDE. Taken together, our studies identify an insulin-degrading enzyme in endosomes which is distinct from IDE. The latter's presence in peroxisomes suggests that its physiological substrate(s) in vivo are polypeptides other than insulin.

Animals↗
PubMed2025

High Prevalence of Potential Molecular Therapeutic Targets in Poorly Differentiated Thyroid Carcinoma.

Poorly differentiated thyroid carcinoma (PDTC) is a rare thyroid cancer with aggressive clinical course and peculiar clinical/pathological characteristics but lacking effective therapeutic options, when surgery is not curative. We aimed at the molecular characterization of PDTC with a specific focus on the identification of potential therapeutic targets. A series of PDTC cases was selected from a multi-institutional network. Fifty-nine samples underwent wide targeted DNA and RNA next-generation sequencing (NGS) testing and immunohistochemical analysis for mismatch repair (MMR) proteins. Gene fusion analysis was enriched by 25 additional samples. Prevalence of MMR protein loss was 11.9%. The most prevalent mutations were in NRAS (25%) and TP53 (25%), mutually exclusive. TERT promoter (TERTp) mutations were detected in 19.6% of cases (10/51). NRAS-mutated cases were enriched for mutations in genes belonging to the same pathway. TP53-mutated samples lacked TERTp co-mutations, but were associated with mutations in PTEN and in genes related to MMR system and/or loss of MMR proteins. TERTp mutations were the most prevalent alterations (28%, 7/25) in a third group that lacked NRAS or TP53 mutations. Four cases harbored gene fusions, including two cases harboring the TBL1XR1::PIK3CA fusion that has never been reported in thyroid cancer, so far. In conclusion, PDTC may be genomically segregated in subgroups with specific molecular characteristics. Overall, targetable gene fusions have a prevalence of 9% (4/42). Moreover, 47% of cases are potential candidates for individualized target therapies since they harbor mutations in genes coding for potentially targetable molecules and/or have defects in the MMR system.

Humans↗
PubMed2003

Xenobiotic response element binding enriched in both nuclear and microsomal fractions of rat cerebellum.

Xenobiotic response element (XRE) is a core nucleotide sequence at the upstream of inducible target genes for the transcription factor aryl hydrocarbon receptor (AhR) that is responsible for signal transduction of exogenous environmental pollutants in eukaryotic cells. Immunoblotting analysis revealed the constitutive expression of AhR-related proteins in rat liver and brain, while specific binding of a radiolabelled probe containing XRE was detected in nuclear preparations of both liver and brain on gel retardation electrophoresis. Among discrete rat brain structures examined, cerebellum exhibited the highest XRE binding with less potent binding in hypothalamus, midbrain, medulla-oblongata, hippocampus, cerebral cortex and striatum. In contrast to liver and hippocampus, cerebellum also contained unusually higher XRE binding in microsomal fractions than that in either nuclear or mitochondrial fractions. Limited proteolysis by V8 protease did not markedly affect XRE binding in cerebellar nuclear extracts, with concomitant diminution of that in hepatic and hippocampal nuclear extracts. In primary cultured cerebellar neurons, indigo was effective in significantly increasing XRE binding only when determined immediately after sustained exposure for 120 min in the presence of high potassium chloride. These results suggest the abundance of as-yet unidentified proteins with high affinity for XRE and responsiveness to indigo in both nuclear and microsomal fractions of rat cerebellum.

Animals↗
PubMed2004

Multiple functionally redundant signals mediate targeting to the apicoplast in the apicomplexan parasite Toxoplasma gondii.

Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle--the apicoplast--acquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP+ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.

Amino Acid Sequence↗
PubMed2001

Activation of Arp2/3 complex-mediated actin polymerization by cortactin.

Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.

3T3 Cells↗
PubMed2005

Dermatan carriers for neovascular transport targeting, deep tumor penetration and improved therapy.

A new approach to functional tumor imaging and deep interstitial penetration of therapeutic agents is to target the upregulated transport activities of neovascular endothelium. Agents are formulated with the anionic glycosaminoglycan, 435-type dermatan sulfate (DS 435, 22.2 kDa), chemically enriched for oligosaccharide sequences that confer high heparin cofactor II binding and correlate with high tumor uptake. A magnetic resonance (MR) imaging agent is prepared as self-assembling, 5-nm nanoparticles of Fe(+3):deferoxamine (Fe:Df) bound by strong ion pairing to DS, which forms the outer molecular surface (Zeta potential -39 mV). On intravenous (i.v.) injection, Fe:Df-DS rapidly (<7 min) and selectively targets and transports at high capacity across the neovascular endothelium of large (2-cm) Dunning prostate R3327 AT1 rat tumors; releases from the abluminal surface, due to reversible binding of its multivalent, low-affinity (K(d) 10(-4) to 10(-5)) oligosaccharide ligands; and progressively penetrates the interstitium from its initial site of high uptake in the well-perfused outer tumor rim, into the poorly perfused central subregion. By gamma camera imaging of (67)Ga:Df-DS, the agent avoids normal site uptake and clears through the kidneys with a t(1/2) of 18 min. A therapeutic formulation of DS-doxorubicin (DS-dox) is prepared by aqueous high-pressure homogenization of the drug and DS 435, which produces 11-nm nanoparticles of doxorubicin cores coated with DS (Zeta potential -39 mV) that are stable to lyophilization. Microscopic analysis of tumor sections 3 h after i.v. injection shows much higher overall tumor fluorescence and deeper matrix penetration for DS-dox than conventional doxorubicin (dox): >75 vs. <25 microm between the nearest microvessels. DS-dox also results in enhanced tumor-cell internalization and nuclear localization of the drug. Therapeutic efficacies in established (250 +/- 15 mg) MX-1 human breast tumor xenografts at maximum tolerated doses (MTDs) are (control vehicle, dox, dox-DS) (a) median days to 7-fold tumor growth: 8.3, 25.6 (p = 0.0007), 43.2 (p = 0.0001); (b) complete 90-day tumor regressions: 0/10, 0/10, 4/10. These results demonstrate the potential to develop a novel class of carbohydrate-targeted neovascular transport agents for sensitive, high-resolution (100-microm) MR imaging and improved treatment of larger sized human tumor metastases.

Angiogenesis Inhibitors↗
PubMed1982

Selective deletion of large segments of Balbiani ring DNA during molecular cloning.

Transcription units in Balbiani ring 1 (BR1) and Balbiani ring 2 (BR2) of Chironomus tenans salivary glands are comprised of about 40 kb of repetitive DNA sequences organized in a satellite-like array. Because of this sequence organization, it was possible to prepare 30 to 40-kb target DNA fragments for cloning by performing limit restriction endonuclease digestion of high-Mr genomic DNA. These fragments were ligated to cohesive termini of the linearized cosmid, pHC79, packaged in vitro, and used to transduce Escherichia coli. Alternatively, target fragments were randomly sheared to a mean length of 8-10 kb, annealed to plasmid pBR322 using homopolymeric tails, and used for bacterial transformation. Recombinant cosmids and plasmids generally contained inserts which were proportional to the length of target fragments used in ligation reactions. However, the subset of recombinants that hybridized to 32P-labeled 75S RNA (highly enriched in BR1 + BR2 sequences) had disproportionately smaller inserts. With the exception of one metastable clone with a 2.1-kb insert, all others had inserts of 0.8 or 0.4 kb. Similar results were obtained in host cells that were recA- or recBC-. The most likely conclusion is that repetitive BR sequences are highly unstable during replication in E. coli and are selectively deleted.

Animals↗
PubMed1999

Use of embryonic and somatic cells for production of transgenic domestic animals.

In contrast to the highly developed genetic modification systems available for manipulating the mouse genome, at this time only simple gain of function modifications can be undertaken in domestic species. Clearly, the greatest barrier to gene targeting in domestic species has been the unavailability of cell lines that can be modified in vitro and still be used to generate a living organism. In the mouse, the embryonic stem (ES) cells and embryonic germ (EG) cells have fulfilled that role. While the nuclear transfer procedures have solved this problem in sheep and cattle, in swine ES and EG cells are still needed. In addition, targeting in domestic species is affected by the need to develop targeting constructs containing isogenic DNA regions. As a result, it is necessary to isolate the gene of interest, sequence required regions, and develop isogenic targeting constructs by technologies such as long-range PCR. On the positive side, enrichment protocols developed in the mouse can be applied to domestic species, thus facilitating the identification of correctly modified cell lines. Hence, progress in mammalian cloning, the development of EG cell lines, and advances in gene targeting presently allows the introduction of precise genetic modifications into the domestic animal genome.

Journal Article↗
PubMed1999

Aptamers: an emerging class of molecules that rival antibodies in diagnostics.

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.

Antibodies↗
PubMed2004

[Novel methods to detect cytokines by enzyme-linked oligonucleotide assay].

The development of the systematic evolution of ligands by exponential enrichment (SELEX) process has made it possible to isolate oligonucleotide sequences with the capacity of recognizing virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are useful as a class of molecules that rival antibodies in diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. To meet the shortcomings of antibodies, aptamers have the following advantages. Aptamer does not depend on animals, cells, or even in vivo conditions and produced by chemical synthesis with extreme accuracy and reproducibility. Once denatured, functional aptamers could be regenerated easily within minutes. They are stable to long-term storage and can be transported at ambient temperature. We describe here an enzyme -linked oligonucleotide assay that use a SELEX-derived RNA aptamer to detect hTNFalpha. In order to protect from nuclease attack, the RNA aptamer was modified by replacement of 2'-NH2 for 2'-OH at all ribo-purines. In a sandwich micro-plate assay, hTNFalpha monoclonal antibody was coated on the surface of the plate, biotin-labeled RNA aptamer was used as a detect molecle. HTNFalpha was diluted by pooled human serum as standard, and streptavidin-horseradish peroxidase-substrate system was added for detection. Accuracy, precision, sensitivity, specificity of ELONA method were analyzed. The levels of hTNF-alpha in normal human serum samples were assayed by the ELONA and the ELISA processes. The resultes demonstrate that a sandwich assay using a SELEX-derived RNA aptamer has parameters for accuracy, precision, sensitivity, specificity well within the limits expected of a typical enzyme-linked assay. There is no significant difference between the results of ELONA and ELISA. The minimum detection level was 100 pg/mL. This method will be useful for detection of almost all the cytokines and other protein molecules.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain.

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.

Biodegradation, Environmental↗
PubMed1992

Targets of homeotic gene regulation in Drosophila.

We have used a chromatin immunopurification approach to identify target genes regulated by the homeotic gene Ultrabithorax. A monoclonal antibody against the Ultrabithorax gene product is used to immunopurify in vivo Ultrabithorax protein binding sites in embryonic chromatin. The procedure gives an enrichment of sequences with matches to a consensus homeodomain binding site. In one case we have shown that an immunopurified sequence lies within a 4 kb fragment that acts in vivo as a homeotic response element. We anticipate that this approach will enable us to identify further targets, allowing the analysis of their regulation and function. The chromatin immunopurification strategy may be of general application for the identification of direct in vivo targets of DNA-binding proteins.

Animals↗
PubMed1988

Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.

Gene targeting--homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences--in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive nd negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt- and int-2- mutants, but it should be applicable to any gene.

Animals↗
PubMed1991

cDNA cloning of a human androgen-induced mRNA exhibiting an early and protein synthesis-independent induction.

The detailed mechanism of action of androgens remains unknown. We have used an androgen-dependent human prostate cancer cell line and a subtractive cDNA hybridisation strategy to enrich for androgen-dependent sequences. This yielded a cDNA clone which exhibits the characteristics of a primary trans-activated target for androgens. This androgen-regulated gene encodes a polyadenylated 4.5 kb mRNA which is induced 30-50-fold within 3 h of treatment with 10 nM dihydrotestosterone. The induction does not require continued protein synthesis as it is maintained in the presence of protein synthesis inhibitors.

Androgens↗
PubMed1993

Interaction between the thyroid hormone receptor and co-factors on the promoter of the gene encoding phospho enol pyruvate carboxykinase.

Using transient transfection studies we localized a thyroid hormone-responsive element on the promoter of the rat phospho-enol pyruvate carboxykinase gene between 355 and 174 bp upstream of the transcription start site. DNAse 1 footprinting analysis within this region showed that a 28 bp fragment at position -324 to -297 was protected by the thyroid-hormone receptor. This receptor was overexpressed in HeLa cells using a vaccinia virus expression vector. DNA-binding assays with this receptor-enriched nuclear HeLa cell extract revealed that only 20% of the thyroid hormone receptor was able to bind the target-sequence with high affinity (4 nM). Titration with nuclear extract from hepatocytes increased the percentage of c-erbA molecules able to bind to this thyroid hormone-responsive element 4-fold without a change in affinity. Our data show that the complex of the thyroid hormone responsive element of the promoter of the phosphoenol pyruvate carboxykinase gene and the thyroid hormone receptor contains only a single receptor molecule suggesting the formation of a heterodimer complex. Accordingly, this thyroid hormone receptor/DNA complex is formed only in the presence of co-factors that are present in limiting amounts in the hepatocyte nucleus.

Animals↗
PubMed2006

Liquid chromatography, electrochromatography and capillary electrophoresis applications of DNA and RNA aptamers.

The development of the systematic evolution of ligands by exponential enrichment (SELEX) process has allowed the isolation of oligonucleotide sequences (referred as aptamers) with the capacity to recognize various classes of target molecules with high affinity and specificity. At the present time, various analytical aptamer-based formats have been developed as suitable tools for the detection and quantification of numerous targets, possessing many potential advantages over the most popular antibody technology. In this review, the aptamer applicability to the separation, purification and quantification of various analytes by liquid chromatography, capillary electrophoresis and electrochromatography is addressed.

Chromatography, Liquid↗