SEARCH · Search PubMed

Results for “Target 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.

SourceResearch connectionFrom yearThrough year

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

At least 1,009 records · Page 56Linked to original sources

PubMed1997

A new approach to gene therapy.

Since 1975, different virus vectors have been developed in order to carry functional genes for gene transfer. However, no successful clinical trials have been reported so far. Recently, a new method for altering a single basepair of target DNA was reported using chimeric RNA/DNA oligonucleotides. The replacement of the single basepair in the target sequence can reach an efficacy of 20%. In patients with hemophilia A or B, the mutations (coagulation factors VIII and IX) are well characterized. The mutation-repair method using chimeric RNA/DNA oligonucleotides could provide an alternative for the treatment of hemophilia. The repaired cells will produce normal protein, like that of non-mutated cells, and the expression of the protein will be stable as long as the repaired cells survive. Clinically, by increasing the concentration of the functional protein (5-10%), it is hoped that a severe phenotype can be converted into a milder phenotype. The high replacement efficacy of the target sequence and the safety of the method make this a likely and promising approach for gene therapy in the future. However, no correction has been detected for the mutations in the coagulation factor genes factor genes factor IX and von Willebrand factor by this method so far.

Animals↗

PubMed1998

Detection and characterization of Helicobacter pylori from patients with gastroduodenal diseases.

Polymerase chain reaction and cytotoxin assays were performed to identify as Helicobacter pylori type I (cagA+/tox+) or type II (cagA-/tox-) 56 (59.6%) strains from 94 patients. Of these patients 64 were affected by nonulcer dyspepsia (NUD), 10 by gastric ulcer (GU), 19 by duodenal ulcer (DU), and 1 by both GU and DU. H. pylori strains were tested for cagA using two sets of primers; target sequences were detected in 40-42/56 (71.4-75%) depending on the set of primers used, while cytotoxin-producing strains (tox +) were 26/56 (46.4%). Tox+ strains were isolated in 13/32 (40.6%), 2/7 (28.6%), and 11/17 (64.7%) in NUD, GU, and DU patients, respectively. However, the different percentage between cagA+ strains from NUD patients (13/32; 40.6%) and patients with ulcerative diseases (13/23; 54.2%) is not statistically significant (p = 0.462). Because the two sets of primers employed for amplification of cagA target sequences give different results, we concluded that cagA alone could not be taken as predictive factor for severity of gastroduodenal disease. It has been found that H. pylori type I is associated with duodenal ulcer disease.

Adult↗

PubMed1999

Comparative modeling of CASP3 targets using PSI-BLAST and SCWRL.

We present results of comparative modeling on 11 targets from the CASP3 experiment. Our methods comprise the following steps: first, PSI-BLAST is used to find homologues of the target sequence in the nonredundant GenBank protein sequence database; second, after several iterations of PSI-BLAST, the resulting profile or position-specific similarity matrix is used to search a database of Protein Databank (PDB) sequences; third, from the list of hits resulting from the PDB search, a parent structure is chosen on the basis of the quality of the alignment and the quality of the experimental structure; fourth, this alignment is adjusted manually whenever insertions or deletions take place in secondary structure regions of the parent; fifth, the backbone is modeled from the parent structure and the alignment; and finally, the program SCWRL is used to replace nonconserved side chains onto the parent backbone given the target sequence. For comparison, we also produced structural models from the unaltered PSI-BLAST alignment, from an alignment from the nonprofile version of BLAST, and from the global sequence alignment program CLUSTAL W. Our results indicate that PSI-BLAST produced considerably better alignments than would be possible with either global or local pairwise sequence alignment algorithms and that manual adjustments were helpful. SCWRL, which uses a backbone-dependent rotamer library to predict side-chain conformations, did well in comparison with other methods used in CASP3.

Algorithms↗

PubMed1991

Regulation of HIV-1 gene expression.

The quantity and quality of HIV-1 gene expression is temporally controlled by a cascade of sequential regulatory interactions. Basal HIV-1 transcription is determined by interaction of cellular regulatory proteins with specific DNA target sequences within the HIV-1 long-terminal repeat. The most notable of these protein:DNA interactions involves NF-kappa B, a transcription factor that plays a pivotal role in the activation of genes important for cellular responses to infection and inflammation. A second level of control involves the virally encoded Tat trans-activator. Tat, in combination with as yet unidentified cellular proteins, activates HIV-1 gene expression through a specific interaction with the viral TAR RNA stem-loop target sequence. A final level of regulation is mediated by the viral Rev protein. Rev acts posttranscriptionally to induce the expression of HIV-1 structural proteins and thereby commits HIV-1 to the late, cytopathic phase of the viral replication cycle. Rev activity appears to require a critical, threshold level of Rev protein expression, thus preventing entry into this late phase in cells that are unable to support efficient HIV-1 gene expression. In total, this cascade of regulatory levels allows the HIV-1 provirus to respond appropriately to the intracellular milieu present in each infected cell. In activated cells, the combination of Tat and Rev can stimulate a very high level of viral gene expression and replication. In quiescent or resting cells, in contrast, these same regulatory proteins are predicted to maintain the HIV-1 provirus in a latent or nonproductive state.

Avian Leukosis Virus↗

PubMed1996

Development of an amplification and hybridization assay for the specific and sensitive detection of Mycoplasma fermentans DNA.

A polymerase-chain-reaction-based detection system for Mycoplasma fermentans was established. The highly conserved tuf gene, which encodes elongation factor Tu of prokaryotes, served as target sequence for the PCR. With two PCR oligodeoxynucleotides, which were selected from M. fermentans specific sequences of the tuf gene, we amplified a 850 base pair DNA fragment. Via the biotin-moiety of one primer the PCR fragments were immobilized on streptavidin-coated microtitre plates. After alkaline denaturation a digoxigenin-labelled M. fermentans specific DNA probe was hybridized to the single stranded immobilized PCR fragment. Detection was performed by addition of an alkaline phosphatase conjugated anti-digoxigenin antibody. 4-methyl-umbelliferyl-phosphate was used as a fluorogenic substrate. Amplification of 10 fg chromosomal target DNA was detected by this 'DNA enzyme immuno assay (DEIA)' technique, corresponding to seven genome copies. Our study supports the presumption that the tuf gene proves to be a suitable target sequence for the PCR based detection of any bacterial species. Furthermore, hybridization of PCR fragments with radio-labelled DNA probes should no longer be necessary, because a very sensitive non-radioactive test system can easily be established with the 'DEIA' technique.

Base Sequence↗

PubMed1996

Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria.

Tim23, an essential component of the protein import machinery of the inner membrane of mitochondria (TIM complex), forms dimers that display a dynamic behavior. Dimer formation is promoted by the membrane potential delta psi. Binding of a matrix targeting sequence to Tim23 triggers dimer dissociation. Monomeric Tim23 is present when a preprotein chain is in transit across the TIM complex. Dimerization of Tim23 is dependent on the second half of its N-terminal hydrophilic domain, which is exposed to the intermembrane space. This segment contains a heptad leucine repeat motif with a predicted capacity for dimer formation. We propose that Tim23 exerts a key function in protein import: Tim23 dimers formed in response to delta psi act as receptors for matrix targeting sequences on the surface of the inner membrane. The ensuring dissociation of Tim23 dimer triggers opening of the TIM channel and insertion of the preprotein.

Amino Acid Sequence↗

PubMed2005

[MGB probes detect Streptococcus mutans and Streptococcus sobrinus in real time].

OBJECTIVE: To detect and distinguish Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) quickly in epidemiology and investigate the distribution of S. mutans in the oral of children with rampant caries. METHODS: Designed minor groove binder (MGB) probes according to the gtf gene of S. mutans and S. sobrinus. Detected 9 reference strains of Streptococcus mutans group by MGB probes in real time and after cultivation. Evaluated the results of these two methods. 92 dental plaques from pre-school children with rampant caries were detected in real time with MGB probes. RESULTS: The primers could amplify the target sequences specificity and distinguished S. mutans and S. sobrinus from each other using MGB probes. Though the fluorescence occurred earlier in S. mutans than in S. sobrinus, they had the same results in nature. In 92 children with rampant caries, the detective ratio of S. mutans was 96.7% and that of S. sobrinus was 32.6%. All the samples which could detect S. sobrinus were positive for S. mutans. CONCLUSION: The primers and probe designed from gtf genes of S. mutans and S. sobrinus can amplify the target sequence and distinguish them from each other in real time.

Child↗

PubMed1997

Implication of a 5' coding sequence in targeting maternal mRNA to the Drosophila oocyte.

Early accumulation of maternal mRNA in one of the cells of the cluster of 16 cystocytes is a critical event in the determination of the Drosophila oocyte. A number of developmentally important mRNAs have been shown to accumulate in the early oocyte. We report here the early expression of the yemanuclein-alpha (yem-alpha) transcript, its accumulation in the germarial oocyte and its dynamic localization in the growing oocyte. We have investigated the mechanisms involved in these processes. Microtubules are likely to be involved in both transport and localization as was shown for other maternal transcripts which behave similarly. However, unlike all the cases reported so far, transport and localization are not dependent on 3'UTR sequences. We show that the 5' coding sequence is necessary for the early accumulation of yem-alpha RNA in the oocyte and for its localization pattern during oogenesis.

Animals↗

PubMed1995

Non-radioactive detection of oligonucleotide probes by photochemical amplification of dioxetanes.

We describe a new method of non-radioactive labelling and detection of oligonucleotide probes. The approach is based on a simple chemical principle. Oligonucleotides labelled with methylene blue (a photosensitizer) are hybridized on a membrane to immobilized DNA target sequences. After hybridization and stringency washing 2(-)[3-(hydroxyphenyl)methoxymethylene] adamantane is added to the membrane and the membrane is irradiated with a tungsten lamp light source through a cut-off filter. Thermally stable dioxetanes are amplified during irradiation at the positions of the labelled probe. These amplified dioxetanes are detected using chemically triggered chemiluminescent decay. Signals are recorded on commercial X-ray film. Detection is possible immediately after the last washing step and a hard copy of the blot is obtained within 1 h. Dependent on the level of the target sequences, the sensitivity of the method allows detection of 0.3 pg single-stranded M13mp18(+) plasmid DNA in dot blots and 75 pg in Southern blots. Additional immunological reaction steps and washing steps with blocking reagents and buffers are avoided. Furthermore, expensive reagents and equipment for physical detection are not necessary. The method might be particularly useful for fast routine analysis in forensic and medical applications. The synthesis of the olefin, conditions of hybridization and the protocol of detection are described in detail.

Adamantane↗

PubMed2003

Use of GC clamps in DHPLC mutation scanning.

OBJECTIVE: Development of a systematic mutation detection assay strategy for denaturing high performance liquid chromatography (DHPLC). DESIGN: Adaptation of Guanine and Cytosine (GC)-clamping from denaturing gradient gel electrophoresis (DGGE) to DHPLC. METHODS: Three target sequences harboring known allelic variants were studied to develop a general DHPLC assay design strategy. These were exon 10 of the human RET (REarranged during Transfection) gene, exon 52 of the mouse Col1a2 gene, and exon 9 of the human FAS (APO-1, CD-95) gene. Available software was used to analyze melting curves and determine assay conditions. GC clamps of 20 bp or 36 bp were added to polymerase chain reaction (PCR) primers to introduce a high melting temperature (T(m)) domain to each of the target molecules. DHPLC was performed under partially denaturing conditions. RESULTS: DHPLC assays of PCR-amplified sequences can be developed using a personal computer. The following three steps allowed for mutation detection in all three targets. The target sequence should have a uniform T(m)GC clamps of length sufficient to introduce a second melting domain with a T(m) > or = 8 degrees above that of the target sequence should be appended to one of the primers. The DHPLC assay should be performed at the highest temperature at which the target sequence is predicted to be > or = 90% double stranded CONCLUSION: Addition of GC-clamps to primers facilitates mutation detection by DHPLC. The theoretical basis for this observation is identical to that underlying the utility of GC-clamps in DGGE.

Animals↗

PubMed1999

Methylation-sensitive restriction endonuclease digestion patterns revealed in Vicia faba L. chromosomes by in situ nick-translation.

Restriction endonucleases sensitive to cytosine methylation (HpaII, MspI and HhaI) and 5-azacitidine were used to study the localization of target sequences in Vicia faba metaphase chromosomes by in situ digestion and radioactive or non-radioactive nick-translation. In control experiments, neither isolated DNA nor chromosomes in situ were digested by HpaII and MspI. Pretreatment with demethylating agent, 5-azacitidine resulted both in increased effectiveness of in situ digestion and nick-translation. In 5-azacitidine-treated material, negative bands in M chromosomes appeared. HhaI cleaved isolated DNA, digested it in situ and gave positive signals as a result of nick-translation procedure in metaphase chromosomes. In S chromosomes containing heterochromatin without target sequences for HpaII and MspI, negative bands were shown after nick-translation. Such heterochromatin contains FokI sequences and in situ nick-translation driven by that restriction enzyme resulted in positive bands.

Azacitidine↗

PubMed2002

Multiplex polymerase chain reaction: a practical approach.

Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features.

Humans↗

PubMed2004

Polymorphisms of microsomal triglyceride transfer protein gene and manganese superoxide dismutase gene in non-alcoholic steatohepatitis.

BACKGROUND/AIMS: The pathogenesis of non-alcoholic steatohepatitis (NASH) is poorly understood. The aim of this study was to examine genetic influences on NASH pathogenesis. METHODS: Blood samples from 63 patients with biopsy-proven NASH and 150 healthy controls were analyzed by the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Two functional polymorphisms were studied: the -493 G/T polymorphism in the promoter of microsomal triglyceride transfer protein (MTP) and the 1183 T/C polymorphism in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD). RESULTS: NASH patients had a much higher incidence of the MTP gene G allele (P=0.001) and of the G/G genotype (P=0.002) compared to the controls. Fat occupied more area in liver lobules and the stage of NASH was advanced in patients with the G/G-genotype than in patients with G/T-genotype (P=0.04). NASH patients also had a higher incidence of the MnSOD T/T genotype (P=0.016). CONCLUSIONS: The G allele in the MTP promoter leads to decreased MTP transcription, less export of triglyceride from hepatocytes, and greater intracellular triglyceride accumulation. The T allele in MnSOD mitochondrial targeting sequence leads to less transport of MnSOD to the mitochondria. Therefore, functional polymorphisms in MTP and MnSOD may be involved in determining susceptibility of NASH.

Adult↗

PubMed2026

Genomic hallmarks of depot medroxyprogesterone acetate-associated meningiomas.

BACKGROUND: Population-based studies have linked progestin exposure to increased meningioma risk. However, the molecular basis of meningiomas associated with depot medroxyprogesterone acetate (DMPA)-a common injectable contraceptive-remains undefined. METHODS: We performed an integrated clinicopathologic and genomic analysis of meningiomas from 10 women with long-term DMPA exposure. Tumors underwent histopathological analysis, targeted sequencing, and DNA methylation profiling. Data were integrated with reference cohorts (Baylor and Heidelberg) and analyzed through classifier assignment, consensus clustering, copy number analysis, differential methylation testing, and dimensionality reduction. RESULTS: Depot medroxyprogesterone acetate-associated meningiomas were all newly diagnosed, World Health Organization grade 1 tumors with a predilection for the anterior and central skull base (n = 6). Nine patients harbored multiple meningiomas. Four experienced regression of untreated meningiomas following DMPA cessation, while 5 demonstrated stabilization. Histopathology demonstrated relative overrepresentation of metaplastic morphology, an uncommon meningioma subtype. All DMPA-associated meningiomas mapped to benign molecular groups, and most exhibited low copy number alteration burden. Targeted sequencing revealed enrichment for TRAF7 mutations (n = 5), with no NF2 mutations detected. Eight tumors shared consensus cluster identity, with cohesive grouping on principal component analysis and t-distributed stochastic neighbor embedding. No differential methylation was identified at the progesterone receptor locus. CONCLUSIONS: Depot medroxyprogesterone acetate-associated meningiomas represent a recognizable phenotype within the broader NF2-wildtype/TRAF7-enriched spectrum of benign meningiomas, characterized by chromosomal stability, a shared methylation profile, tumor multiplicity, and regression or stabilization following DMPA cessation. While derived from a small single-institution cohort, these findings provide a molecular framework for understanding progestin-associated meningioma biology, reinterpreting epidemiologic literature, and informing population-level risk stratification.

Humans↗

PubMed2025

Targeted Gene Sequencing in a Male Adult Diagnosed With X-Linked Osteoporosis Due to a Novel p.(Arg398Profs*2) PLS3 Variant.

Pathogenic loss-of-function variants in the plastin-3 gene (PLS3), encoding plastin-3 protein, are associated with early-onset X-linked osteoporosis. We present the case of a young adult male patient, with a history of multiple fragility fractures and blue sclerae, who was clinically diagnosed with osteogenesis imperfecta (OI) type 1 in childhood. He has been managed with intravenous bisphosphonate therapy, leading to an increase in bone density at the spine, stable bone density at the femoral neck, and a period free of fractures while on antiresorptive therapy. Two decades later, with a focus on reproductive family planning, a novel PLS3 variant was identified on genetic testing. This case report highlights an important role for genetic testing in patients with early-onset osteoporosis or a clinical diagnosis of OI. With the emergence of new targeted therapeutics and advanced reproductive options, such as preimplantation genetic testing, obtaining an accurate molecular diagnosis is key.

PLS3↗

PubMed1993

PMLRAR homodimers: distinct DNA binding properties and heteromeric interactions with RXR.

Fusion proteins (named PMLRAR) between PML and the retinoic acid receptor alpha (RAR alpha) are generated as a result of the t(15;17) chromosomal translocation found in acute promyelocytic leukemia (APL). We show here that PMLRAR proteins exist in solution as stable homodimers whose formation is mediated by a presumptive coiled coil in the PML moiety. In contrast to RAR alpha, which requires heterodimerization with RXR for efficient DNA binding, PMLRAR homodimers can bind to target sequences in the absence of RXR, and the binding pattern of PMLRAR homodimeric complexes to directly repeated motif (DR) response elements with 1-5 bp spacers is different from that of RAR/RXR heterodimeric complexes. We show that the presence of RXR induces the formation of PMLRAR/RXR heteromeric complexes which bind to DNA via one RAR DNA binding domain (DBD) and one RXR DBD, like 'classical' RAR/RXR heterodimers. PMLRAR interaction with RXR occurs in solution and in transfected cultured Cos cells, and PMLRAR is able to sequester RXR efficiently in the cytoplasm, suggesting that dominant 'inactivation' of RXR may be a possible mechanism of action for PMLRAR. Accordingly, we show that PMLRAR can both prevent the binding of the vitamin D3 receptor (VDR) to a target sequence in vitro and inhibit vitamin D3-dependent activation of a VDR-responsive reporter gene in transfected cells. These results suggest that both the distinct DNA binding properties of PMLRAR homodimers and the sequestration of RXR by PMLRARs may contribute to the molecular mechanisms which underlie the pathogenesis of APL. We also report that RXR alpha transcripts are down-regulated by RA-treatment in promyelocytic cells.

Amino Acid Sequence↗

PubMed1981

Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. II. Catalytic properties, substrate specificity, and mode of action.

The properties of two DNA methyltransferases, termed M. BsuRIa and M. BsuRIb, whose isolation was described in the preceding paper (Günthert, U., Freund, M., and Trautner, T. A. (1981) J. Biol. Chem. 256, 9340-9345) were compared. Both enzymes recognize the same target sequence in double-stranded DNA, leading to methylation of the internal cytosine: 5'GGCC. The enzymes have identical reaction constants with their substrates, DNA (km = 2.7 nM for the 5' GGCC sequence), and S-adenosyl-L-methionine (km = 0.7 microM). Initial rates of methyl group transfer were proportional to enzyme concentration over a range of 50-fold, indicating absence of aggregation. The enzymes are different in their ionic strength requirements using Tris-HCl, pH 8.4. M. BsuRIa is most active at 100 mM, M. BsuRIb at 440 mM. As measured by incorporation kinetics and heat inactivation, M. BsuRIa is the more stable enzyme of the two. Equilibrium dialysis was used to study the mode of methyl group transfer to the DNA with either enzyme. The data indicate that initially S-adenosyl-L-methionine binds to methyltransferase. This complex attaches to either modified or nonmodified DNA. The methyl group will then be transferred to a nonmodified target sequence, leading to the dissociation of enzyme and S-adenosyl-L-homocysteine from the DNA.

Bacillus subtilis↗

PubMed2002

Specificities and functions of the recA and pps1 intein genes of Mycobacterium tuberculosis and application for diagnosis of tuberculosis.

The worldwide recrudescence of tuberculosis and the widespread appearance of antibiotic resistance have strengthened the need for rapid and specific diagnostic tools. The prevailing microbiological identification of Mycobacterium tuberculosis, the causative agent of tuberculosis, which implies the use of in vitro cultures and acid-fast staining microscopy, is time-consuming. Detection of M. tuberculosis directly in clinical samples through PCR amplification of mycobacterium-specific genes, designed to shorten diagnostic delay, demonstrated reliability and high sensitivity. However, the quality of the diagnosis depends on the specificity of the target sequence for M. tuberculosis complex strains. In the present study, we demonstrated the specificity of recA and pps1 inteins for this complex and thus the feasibility of using intein-coding sequences as a new target for PCR diagnosis. Indeed, the recA and pps1 genes of 36 clinical isolates of M. tuberculosis and 10 field strains of M. bovis were found to be interrupted by an intein sequence at the RecA-a and Pps1-b sites, respectively, while a large number of nontuberculous mycobacterial species failed to demonstrate these insertions. Besides, the MtuPps1, which was cloned and expressed in Escherichia coli, was shown to possess an endonuclease activity. The intein cleaves the 40-bp sequence spanning the intein insertion site Pps1-b in the inteinless pps1 gene. In addition to the PCR amplification of recA and pps1 intein genes as a tool for diagnosis, the specific endonuclease activity could represent a new molecular approach to identify M. tuberculosis.

Bacterial Proteins↗