Search PubMed⌕ Search

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.

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

At least 811 records · Page 45Linked to original sources
PubMed2004

Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus.

A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442-1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin-streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture.

Biosensing Techniques↗
PubMed1999

Sequence to structure alignment in comparative modeling using PrISM.

PrISM (Protein Informatics System for Modeling) is a protein analysis and modeling system in which informatics, alignment, modeling, and assessment modules are integrated in a computational environment where protein analysis and modeling protocols can be designed and assessed interactively. It can then be used automatically and repetitively in response to a variety of protein analysis and modeling problems. PrISM was used to predict a single model for each of the 43 targets in the CASP3 experiment. In this paper, we present results for 13 target sequences, which we consider to be comparative modeling targets with clearly related structural templates. We emphasize the problem of aligning a target sequence to a template structure with various alignment methods. When more than one alignment method and/or parameter set are applied, the final alignment is chosen on the basis of a model ranking system also used in PrISM's fold recognition module. Advanced sequence-template alignment procedures in PrISM are useful in some cases when standard pairwise dynamic programming algorithm fail to make any reasonable global alignment. The same procedures, however, failed in other cases, corresponding to remotely related query-template pairs that involved extensive insertions and deletions.

Algorithms↗
PubMed1991

Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications.

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies--examining the critical parameters and variations and their widespread applications--giving the strengths and limitations of these methodologies.

Bacterial Infections↗
PubMed2005

DNA-arrays with electrical detection: a label-free low cost technology for routine use in life sciences and diagnostics.

Fast and highly parallel DNA analysis are essential for improved biomedical research and development. Currently fluorescence-based methods are state of the art in DNA microarray analysis. The necessity to modify the target DNA with labels is costly, laborious and requires skilled personnel. Moreover, false positive calls from unspecific adsorption are possible and it is difficult to discriminate perfect matching target sequences from those with a single mismatch. In this paper a new and simple electrochemical approach for hybridisation detection without the need of labelling the target DNA is described. The EDDA (Electrically Detected Displacement Assay) method uses a solution of short redox-labelled signalling oligonucleotides (oligonucleotides carrying a covalently attached redox active compound like ferrocene) to characterize the hybridisation state of label-free capture probe DNA immobilised on gold electrodes. The number of capture probes associated with signalling oligonucleotides is determined by chronocoulometry. This technique allows to separate the electrochemical response of capture probe associated signal probes from the response of freely diffusing signalling probes. In the absence of the complementary target sequences the redox-labelled signalling probes at the surface give rise to an instantaneous increase of the detection signal, while freely diffusing signalling probes show a significantly delayed response. Hybridisation with targets complementary to the capture probe displace the loosely associated signalling probes thereby decreasing the instantaneous signal. Besides an introduction to the EDDA technology, data validating the method for biological material will be presented and an outlook to the detection of single nucleotide polymorphisms (SNPs) is given.

Biosensing Techniques↗
PubMed2004

DNA displacement assay integrated into microfluidic channels.

This paper describes the development of a unique fluorescence-based DNA diagnostic microfluidic assay that does not require labeling of the target sequence prior to analysis. The assay is based on the displacement of a short sacrificial fluorescent-tagged indicator oligomer by a longer untagged target sequence as it is electrophoresed through a DNA-containing hydrogel plug immobilized in a microfluidic channel. The distinct advantages of this assay are the short sensing times, as a result of directed electrophoretic transport of target DNA to the sensing element, combined with the ability to detect nonlabeled target DNA.

DNA↗
PubMed1988

Theoretical analysis of 'addressed' chemical modification of DNA.

Chemical "addressed" modification of DNA involves treatment of single-stranded DNA with oligonucleotides complementary to certain target sequences in this DNA and bearing a groupings reactive towards DNA bases. The binding of oligonucleotides can occur both at completely (specific) and incompletely (nonspecific) complementary sites. We analyse the modification of a fragment that is flanked by two target sequences complementary to a given oligonucleotide address, contains no more such targets and has some randomly distributed sites for nonspecific binding. Conditions for the maximum ratio between specific and non-specific modification are determined. We find the probability of both target termini being specifically modified without any non-specific modification occurring within the fragment up to a given moment in time. Quantitative analysis is based on the use of known features of the specific and non-specific binding of an oligonucleotide to DNA sites. This analysis shows the possibility of specific cutting of DNA based on addressed modification.

Base Composition↗
PubMed1998

A subtractive hybridisation method for the enrichment of moderately induced sequences.

Moderately induced genes often escape detection in conventional subtraction hybridisation cloning. Here a modification of a phagemid subtraction protocol is described that overcomes this problem. The protocol uses low ratio hybridisation of driver to target sequences to allow enrichment of the sequences of interest, and back-hybridisation of the subtracted sequences with induced sequences to reduce the accumulation of false positive clones. The procedure takes advantage of the quantitative representation of cellular RNA populations in cDNA libraries, therefore, they may serve not only as renewable sources of driver and target sequences, but also as sources of population cRNAs used in northern blots and differential Southern blots.

Animals↗
PubMed2005

Targeting and regulation of the HER-2/neu oncogene promoter with bis-peptide nucleic acids.

Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.

Alkylating Agents↗
PubMed2002

Evolution of Ig DNA sequence to target specific base positions within codons for somatic hypermutation.

Ig variable (V) region genes are subjected to a somatic hypermutation process as B lymphocytes participate in immune reactions to protein Ags. Although little is known regarding the mechanism of mutagenesis, a consistent hierarchy of trinucleotide target preferences is evident. Analysis of trinucleotide regional distributions predicted and we now empirically confirm the surprising finding that the framework 2 region of kappa V region genes is highly mutable despite its importance to the structural integrity and function of the Ab molecule. Interestingly, much of this mutability appears to be focused on the third codon position where synonymous substitutions are most likely to occur. We also observed a trend for high predicted mutability for codon positions 1 and 2 in complementarity-determining regions. Consequently, amino acid replacements should occur at a higher rate in complementarity-determining regions than in framework regions due to the distribution and subsequent targeting of microsequences by the mutation mechanism. Our results reveal a subtle tier of V region gene evolution in which DNA sequence has been molded to direct mutations to specific base positions within codons in a manner that minimizes damage and maximizes the benefits of the somatic hypermutation process.

Animals↗
PubMed1994

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana.

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)(+)-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses. The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5' non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5' sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3' sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site. This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.

Amino Acid Sequence↗
PubMed2003

Identification of Drosophila MicroRNA targets.

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential, as it allows reduction of the sequence search space. Improvements in genome annotation and increased availability of cDNA sequences from other genomes will allow more sensitive screens. An increase in the number of confirmed targets is expected to reveal general structural features that can be used to improve their detection. While the screen is likely to miss some targets, our study shows that valid targets can be identified from sequence alone.

3' Untranslated Regions↗
PubMed1998

Quantitation of dopamine D2 receptor mRNA in a mesencephalic cell culture using a nonradioactive competitive reverse transcription polymerase chain reaction method.

Studies on gene expression during differentiation and maturation processes have to cope with determinations of extremely low steady state levels of specific mRNA. Using the experimental model of dopamine D2 receptor (D2R) expression in a primary mesencephalic cell culture we worked out a quantitative reverse transcription polymerase chain reaction method which allows to analyze and quantify mRNA levels of cells present in a few wells of the culture. The method uses an internal cRNA standard which shares both primer binding sites and PCR product length with the target sequence. The amplicons are quantitated in microplates by hybridization with immobilized capture probes that allow for the distinction of internal standard and target sequences followed by the chemiluminescent detection of hybridized DNA. Applying this method the levels of D2 receptor mRNA of the mesencephalic cell culture on day in vitro 1 amounted to about 250 fg/microgram RNA and increased to about 1200 fg/microgram RNA on day in vitro 13-15.

Animals↗
PubMed1990

Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides.

Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.

Animals↗
PubMed1997

Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi.

This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50,066. Comparison of the deduced amino acid sequence of LipDH from T. cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively. An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd1 of T. cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N-terminus of the mature protein. This system allowed the synthesis of 1000 U T. cruzi LipDH/1 bacterial cell culture. The recombinant protein was purified to homogeneity by (NH4)2SO4-precipitation and affinity chromatography on 5' AMP-Sepharose. The K(m) values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite. LipDH is present in all major developmental stages of T. cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite. In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason T. cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystallization of the protein.

Amino Acid Sequence↗
PubMed2004

Detection of low copy numbers of HIV-1 proviral DNA in patient PBMCs by a high-input, sequence-capture PCR (Mega-PCR).

An internally controlled high-input PCR method, termed HIV-1 Mega-PCR was developed to lower the detection limit of HIV-1 DNA polymerase chain reaction (PCR) and to improve its value as a complementary diagnostic test. It is based on PCR amplification of two target sequences in the gag gene of HIV-1 following the selective capture of the targeted sequence and removal of unselected DNA from up to 500 microg of DNA. Efficient selection and amplification was monitored by inclusion of two mimic plasmids. The method was evaluated with buffy coat cells from healthy blood donors which were spiked with blood from 106 different HIV-1-infected individuals, and with 107 HIV-1 seronegative control buffy coats. All specimens from HIV-infected individuals were positive by a PCR protocol using 1 microg of patient DNA. Amplification of 1 microg DNA of the 106 spiked, diluted samples resulted in 68 double positive, 14 single positive, and 24 double negative reactions. In the Mega-PCR, the average input was 260 +/- 84 microg DNA containing an estimated 1.1 +/- 0.6% of spiked patient DNA. Of the 106 samples tested by Mega-PCR, 102 were positive and three negative. One failed to select the mimic plasmid. Among the 107 negative buffy coat controls, none was false-positive and four exhibited a failure of the internal reaction control. Application of HIV-1 Mega-PCR to clinical specimens from seroreverting newborns of HIV-infected mothers and seroindeterminate, PCR-negative specimens revealed no indication for HIV infection, whereas three samples from confirmed, HIV-1-infected but PCR negative individuals showed evidence of the presence of HIV-1 DNA. Mega-PCR lowers the detection limit of an individual analysis to approximately 0.01 HIV-1 DNA copies/microg of applied DNA and may help to confirm or exclude HIV-1-infection in difficult situations diagnostic.

Adult↗
PubMed1989

Determinant differences between the rabbit and mouse immunoglobulin kappa enhancers impair the activity of the rabbit enhancer in mouse myeloma cells.

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.

Animals↗
PubMed2002

Critical role of the pleckstrin homology and cysteine-rich domains in Vav signaling and transforming activity.

Vav family proteins are members of the Dbl family of guanine nucleotide exchange factors and activators of Rho family small GTPases. In addition to the Dbl homology (DH) domain important for guanine nucleotide exchange factor catalytic function, all Dbl family proteins contain an adjacent pleckstrin homology (PH) domain that serves to regulate DH domain activity. Although the role of the PH domain in Vav function has been evaluated extensively, its precise role and whether it serves a distinct role in different Vav proteins remain unresolved. Additionally, the precise role of an adjacent cysteine-rich domain (CRD) in regulating DH domain function is also unclear. In this study, we evaluated the contribution of these putative protein-protein or protein-lipid interaction domains to Vav signaling and transforming activity. In contrast to previous observations, we found that the PH domain is critical for Vav transforming activity. Similarly, the CRD was also essential and served a function distinct from that of the PH domain. Although mutation of either domain reduced Vav membrane association, addition of plasma membrane targeting sequences to either the CRD or PH domain mutant proteins did not restore Vav transforming activity. This result contrasts with other Dbl family proteins, where a membrane targeting sequence alone was sufficient to restore the loss of function caused by mutation of the PH domain. Furthermore, green fluorescent protein fusion proteins containing the PH domain or CRD, or both, failed to target to the plasma membrane, suggesting that these two domains also serve regulatory functions independent of promoting membrane localization. Finally, we found that phosphatidylinositol 3-kinase activation may promote Vav membrane association via phosphatidylinositol 3,4,5-triphosphate binding to the PH domain.

3T3 Cells↗
PubMed1993

PCR amplification using a single cell allows the detection of the mtDNA lesion associated with Leber's hereditary optic neuropathy.

The development of the polymerase chain reaction (PCR), which routinely can amplify specific target sequences more than one billion-fold, has made it possible to produce readily detectable amounts of DNA from a few copies of very rare sequences. We have begun a study of mitochondrial myopathies with the purpose of developing a diagnostic test using PCR to amplify appropriate mitochondrial DNA (mtDNA) target sequences from small amounts of sample. We have developed a 15-min procedure for recovering mtDNA which can be amplified by PCR to detectable levels, from as little as 30 microliters of blood or 5 microliters of amniotic fluid. We have microscopically selected HL60 cells, and have found that 28 cycles of PCR allows the detection of mitochondrial targets from a single cell. Using micromanipulation techniques, we utilized this approach to analyze mtDNA from a single cell isolated from an 8-cell stage mouse blastocyst. Finally, a single cell cultured from a patient with Leber's hereditary optic neuropathy, a mitochondrial myopathy, provided sufficient mtDNA for detection of the single base substitution that leads to loss of a restriction endonuclease recognition site for SfaNI and generation of a site for MaeIII.

Animals↗