Gene/Protein Disease Symptom Drug Enzyme Compound
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Some of the most polymorphic short tandem repeats (STRs) have alleles differing in size by as little as one basepair. Since sizing precision with commercially available internal standards ordinarily does not allow safe discrimination at this level, typing is accomplished through comparisons with allelic ladders run on each gel. Here we introduce the use of optimally spaced sequenced alleles as a dedicated internal standard for measurement and typing of hyperpolymorphic STRs. We have constructed such a dedicated internal standard for HUMACTBP2 ([1]; Moos and Gallwitz, EMBO I., 5 (1983) 757-761) typing, including 25 sequenced, ROX labelled HUMACTBP2 alleles spanning the size range for alleles at this locus (233-333 basepairs (bp)). Comparisons of inter-gel runs of selected alleles with this dedicated standard and the commercial GS500 internal standard demonstrate that only the presently described internal standard is suited for direct typing based on fragment length measurements only. Obvious advantages of this typing procedure are that fragment measurements are precise and in accordance with sequenced lengths, and that the typing procedure via an external allelic ladder is avoided. Sequences of the alleles used in the internal standard as well as of selected alleles for allelic ladders in two other hyperpolymorphic AAAG-repeat STRs, D11S554 (Phromchotikul et al., Hum. Mol. Genet., 3 (1991) 21) and HUMAPOA11 (Kimpton et al., PCR Methods Appl., 3 (1993) 13-22) are also presented. To allow fragment length analysis of these two STRs, five D11S554 alleles, spanning from 176 to 225 basepairs, were added to the dedicated internal standard. Performing similar comparisons as for HUMACTBP2, it is demonstrated that even if sizing accuracy is not as good as with HUMACTBP2, typing based on measured fragment size only may be achieved with both other STRs as well. Selecting different colours for the three STRs, triplex electrophoretic runs were performed of a Norwegian population database of 300 unrelated individuals. The probability that two unrelated individuals have the same type in all three STRs is 5 x 10(-7), and the combined paternity exclusion power 99.77%, indicating that this STR selection may represent a good choice for forensic genetic casework.
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PMID:A dedicated internal standard in fragment length analysis of hyperpolymorphic short tandem repeats. 918 30

This article describes a procedure for modification of the commercially prepared GeneScan 2500 size standard for allelotyping with large DNA fragments such as variable number tandem repeats (VNTRs). Here a procedure was used to adapt commercially available size standards for the sizing of the interleukin-6 (IL6) 3'VNTR, which has allele sizes ranging from 600 to 900 base pairs. The procedure involves inclusion of products from the target PCR reaction as additional size standards for use with the size standard and is therefore applicable to the sizing of any product with any commercial size standard. Initially alleles were sized by fluorescent cycle sequencing to give a true estimate of their size (base pairs). Subsequently, the major alleles were labeled with a pig-tailed ROX-dye-labeled primer and inserted into the standard range. Finally alleles were resized following PCR with an un-pig-tailed HEX labeled primer and the modified standard. Once the size standard is modified, stocks can be stored indefinitely and replenished by re-PCR of selected alleles using the ROX-labeled primer. Modification of this approach can be applied to the sizing of any product where it is thought that commercially available size standards are performing suboptimally. Modification of size standards in this way does not affect performance in size regions other than those for which the extra products are included.
Mol Biotechnol 1999 Dec 15
PMID:Modification of the GeneScan 2500 fluorescent dye standard for accurate product sizing. 1093 31

Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.
Mol Cell Probes 2005 Feb
PMID:Development of a novel internal positive control for Taqman based assays. 1565 20

Selection of optimal reaction master mix reagents is essential to obtain the best performance with diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Every year the number of commercially available master mix kits increases, so it is prudent to periodically evaluate kits on the market. In this study we evaluated five commercial real-time RT-PCR master mix kits, the RealMasterMix RT-PCR ROX kit, the AgPath-ID One-Step RT-PCR kit, the SuperScript III Platinum One-step Quantitative RT-PCR system, the QuantiTect Probe RT-PCR kit, and the LightCycler RNA HybProbe amplification kit, using a 5'nuclease assay for Ebola virus. The kits were evaluated using the manufacturer's recommended conditions, as well as conditions which have been used with the Ebola virus assay during outbreaks. When evaluated for use in Ebola virus RNA detection, the AgPath-ID kit resulted in the greatest sensitivity in comparison to the other four kits. The efficacy of the AgPath-ID kit was instrument-independent in the five real-time PCR platforms tested. This study demonstrated that Ebola virus RNA detection was not equivalent among the master mix reagents studied and, thus, that this variable can affect real-time RT-PCR assay sensitivity. Furthermore, this study rates the master mix reagents for their suitability, providing diagnostic laboratories the option to select from these kits to suit their specific laboratory needs for real-time RT-PCR.
Mol Cell Probes 2010 Dec
PMID:Cross-platform evaluation of commercial real-time reverse transcription PCR master mix kits using a quantitative 5'nuclease assay for Ebola virus. 2073 12

In order to prepare antibacterial and radio-opaque dental resin, a methacrylate monomer named 2-Dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) with both antibacterial and radio-opaque activities was added into a 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropyl)-phenyl]propane (Bis-GMA)/methyl methacrylate (MMA) dental resin system. Degree of conversion (DC), flexural strength (FS) and modulus (FM), water sorption (WS) and solubility (WSL), antibacterial activity, and radio-opacity (ROX) of the obtained dental resin system were investigated. Bis-GMA/MMA resin system without DDMAI was used as a control. The results showed that DDMAI could endow BIS-GMA/MMA resin system with good antibacterial (p < 0.05) and radio-opaque function without influencing the DC (p > 0.05). However, incorporating DDMAI into Bis-GMA/MMA resin could reduce mechanical properties (p < 0.05) and increase WS and WSL (p < 0.05), thus further work is needed in order to optimize the resin formulation.
Int J Mol Sci 2013 Mar 07
PMID:Preparation and evaluation of dental resin with antibacterial and radio-opaque functions. 2347 Sep 23

The formation of axillary meristems in leaf axils is a prerequisite for the development of lateral shoots, which largely contribute to plant architecture. Several transcription factor-encoding genes, including CUC3, RAX, LAS, LOF1, and ROX, have been cloned by screening for axillary meristem mutants in Arabidopsis thaliana. These genes will facilitate our understanding of the mechanisms underlying axillary meristem development. In this study, we report the cloning of five genes from cotton (Gossypium hirsutum L.) that are orthologous to A. thaliana REGULATORS OFAXILLARY MERISTEMS (RAX) and tomato Blind (Bl), and they are designated GhRAX1, 2, 3, 4, and 5. Sequence analyses indicated that all five genes shared conserved protein domains with RAX and Bl. Phylogenetic analyses of protein sequences revealed that GhRAX2/3/4 were close to RAX1, whereas GhRAX1 and GhRAX5 were close to RAX3. Expression patterns of these genes in different tissues were also analyzed using real-time PCR.
Genet Mol Res 2015 Oct 05
PMID:Molecular cloning and expression analysis of five GhRAXs in upland cotton (Gossypium hirsutum L.). 2650 59

Nogo-B and its receptor (NgBR) are involved in blood vessel growth in developing lungs, but their role in pulmonary artery smooth muscle cell (PASMC) growth is unknown. We hypothesized that NgBR regulates growth of PASMCs by modulating the function of endoplasmic reticulum (ER) and formation of reactive oxygen species (ROS). In utero constriction of the ductus arteriosus created pulmonary hypertension in fetal lambs (hypertensive fetal lamb [HTFL]). PASMCs isolated 8 days after surgery were assessed for the alteration of protein levels by immunoblots and ROS formation by dihydroethidium and Cell ROX deep red fluorescence. NgBR small interfering RNA and plasmid DNA were used to manipulate NgBR levels. Proliferation and wound healing were assessed by cell counts and scratch recovery assay, respectively. Acute ER stress was induced by tunicamycin. Differences of mitogen-activated protein kinase and Akt pathway activation in HTFL versus control PASMCs were evaluated. Results showed that HTFL PASMCs had decreased NgBR levels and increased proliferation, wound healing, ER stress, and ROS formation compared with controls. Knockdown of NgBR in control PASMCs generated a phenotype similar to HTFL, and overexpression in HTFL restored the defective phenotype to control. Decreased NgBR levels were associated with increased ROS formation in HTFL PASMCs. Subsequently, scavenging ROS decreased proliferation and wound healing. Mechanistically, ROS formation decreases NgBR expression, which induces ER stress. This leads to extracellular signal-regulated kinase pathway activation and PASMC phenotype alteration. Our data suggest that decreased NgBR expression in pulmonary hypertension of the newborn contributes to increased PASMC proliferation and oxidative stress, which lead to the pathogenesis of lung injury.
Am J Respir Cell Mol Biol 2016 06
PMID:Nogo-B Receptor Modulates Pulmonary Artery Smooth Muscle Cell Function in Developing Lungs. 2665 54

Accurate monitoring of low levels of viral load (the number of viral particles per milliliter of plasma) in HIV-infected patients is important in terms of evaluation of the progress of antiretroviral therapy. The general approach for detection of low copy HIV RNA is reverse transcription combined with quantitative real-time PCR based on fluorescence detection. The selection of primers and the structure of fluorogenic oligonucleotide probes are crucial for sensitivity and accuracy of the assay. In this chapter, we report the RT-qPCR protocol for detection of low copy HIV RNA using double stranded Yin-Yang DNA probes containing identical fluorescent dyes on each strand of the probe. Dye residues attached to the 3'-end of an oligonucleotide and 5'-end of the complementary oligonucleotide form a self-quenched aggregate in a Yin-Yang duplex probe, and display fluorescence light up upon probe strand displacement with the target sequence amplified in the course of PCR. Among several fluorescent dyes tested (R6G, ROX, Cy5) the ROX labeled Yin-Yang probes showed better fluorescence increase and lower Ct values. All the homo Yin-Yang probes were superior to corresponding dye-quencher probes and allowed reliable detection of 10-10,000 copies of HIV RNA per mL.
Methods Mol Biol 2020
PMID:RT-qPCR Detection of Low-Copy HIV RNA with Yin-Yang Probes. 3166 60