Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR.
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Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR.
Viral hemorrhagic fevers (VHFS) are acute infections with high mortality rates. Significant VHF agents are Ebola and Marburg viruses (MBGV / EBOV), Lassa Virus (LASV), Crimor Congo haemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), viruses dengue (DENV) and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and distinguish; Fast and reliable laboratory diagnosis is required in suspect cases. We have set up six in a single step and real-time reverse-PCR transcription tests for these pathogens based on the transcriptase-platinum taq polymerase taq.
NEW-primer detection probes and / or 5′-nucleases have been designed for RVFV, DENV, YFV and CCHFV using the latest DNA database entries. PCR products have been detected in real time on a lightcycler instrument using 5′-nuclease technology (RVFV, DENV and YFV) or Sybrgreen (MBGV / EBOV, LASV and CCHFV) dye intercalation). The Sybrgreen inhibitor effect on the opposite transcription has been surmounted by an initial immobilization of the dye in the reaction capillaries. The universal cycling conditions for the detection of Sybrgreen and 5′-nuclease probe have been established. Thus, up to three tests could be carried out in parallel, facilitating rapid tests for several pathogens. All tests have been carefully optimized and validated in terms of analytical sensitivity using Vitro transcribed RNA.
The detection limits >> or = 95% determined by a probity regression analysis ranged from 1,545 to 2,835 equivalents of viral genome / ml serum (8.6 to 16 copies of RNA per test). The adequacy of the tests was illustrated by the detection and quantification of the viral RNA in serum samples of VHF patients.
Mutagenesis directed by the double-stranded DNA site by the polymerase chain reaction.
We have developed an easy procedure for the mutagenesis directed by the rapid PCR site of the DNA with double-border. The increase in the initial concentration of template and the decrease of PCR cycles to 5-10 allows us to reduce the rate of mutations of second undesirable site and considerably increase time savings. After PCR, the DPNI treatment is used to select against parental DNA molecules. The DPNI (target sequence 5′-GM6ATC) is specific to the methyl and hemimethyl DNA and is used to digest the parental DNA and select for the amplified DNA containing mutations. DNA isolated from almost all current strains Escherichia coli is the methyl dam and therefore sensitive to diNI digestion. The PFU DNA polymerase is used before the intramolecular ligation of the linear template, to remove extended bases on the 3 ‘ends of the PCR product by Taq DNA polymerase.
The recirculated vector DNA incorporating the desired mutations is transformed into E. coli. This method can be used independently of any host strain and vector. The contribution of PCR artifacts to the diversity of the ARRN genes sequence of a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be forced by grouping the 99% sequence similarity group sequences. Other artifacts (chimeras and heterodeplex molecules) were significantly reduced using modified amplification protocols. Surprisingly, no falsity of sequence types have been detected in the two libraries built from amplified PCR products for different cycle numbers. Recommendations for modifying the amplification and reporting protocols of 99% diversity estimates of sequence similarity as a standard are given.
Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR.
Enzymatic assembly of overlapping DNA fragments.
Three methods of assembling several DNA molecules overlapping are described. Each method shares the same basic approach: (i) an exonuclease eliminates the nucleotides of the ends of the dual-fired DNA molecules (DS), exposing specific advertising DNA (SS) overhangs; (ii) SSDNA gaps of the joined molecules are filled by DNA polymerase and pseudonyms are sealed covalently by DNA ligase. The first method uses the 3′-exonuclease of T4 DNA polymerase (T4 POL), Taq DNA polymerase (Taq Pol) and the Taq Taq Ligase (Taq Lig) in a two-step thermocyclic reaction. The second method uses 3′-exonuclease III (EXOIII), Taq Pol and Taq Antibody Lag in a thermocyclic reaction in one step.
The third method uses the 5′-T5 exonuclease, the polymerase of the DNA phusion® and the TAQ LIG in an isothermal reaction in one step and can be used to assemble both SSDNA and DSDNA. These assembly methods can be used to transparently construct synthetic and natural genes, genetic pathways and integer genomes and could be very useful for molecular engineering tools. The nucleotide sequences in three hypervarial regions of the Human Immunodeficiency Virus (HIV-1) will envisage by provirus sequencing present in peripheral blood mononuclear cells of individuals infected with HIV. Simple molecules of target sequences have been isolated by limiting dilution and amplified in two steps by reaction of the polymerase chain, using nested primers.
Description: DNA-directed RNA polymerase II subunit RPB1, also known as RPB1, is an enzyme that in humans is encoded by the POLR2A gene. It is mapped to 17p13.1. This gene encodes the largest subunit of RNA polymerase II, the polymerase responsible for synthesizing messenger RNA in eukaryotes. The product of this gene contains a carboxy terminal domain composed of heptapeptide repeats that are essential for polymerase activity. These repeats contain serine and threonine residues that are phosphorylated in actively transcribing RNA polymerase. In addition, this subunit, in combination with several other polymerase subunits, forms the DNA binding domain of the polymerase, a groove in which the DNA template is transcribed into RNA.
Description: RNA polymerase II (Pol II) is an enzyme that is composed of 12 subunits and is responsible for the transcription of protein-coding genes. Transcription initiation requires Pol II-mediated recruitment of transcription machinery to a target promoter, thereby allowing transcription to begin. The largest subunit of Pol II (referred to as RPB1 or RPB205) is a 1,840 amino acid protein that contains one C2H2-type zinc finger and a C-terminal domain comprised of several heptapeptide repeats. Although Pol II function requires the cooperation of all twelve subunits, the largest subunit conveys Pol II catalytic activity and, together with the second largest subunit, forms the active center of the Pol II enzyme. Additionally, the large subunit participates in forming the DNA-binding domain of Pol II, a groove that is necessary for transcription of the DNA template. Without proper function of the large subunit, mRNA synthesis and subsequent transcription elongation cannot occur.
Description: RNA polymerase II (Pol II) is an enzyme that is composed of 12 subunits and is responsible for the transcription of protein-coding genes. Transcription initiation requires Pol II-mediated recruitment of transcription machinery to a target promoter, thereby allowing transcription to begin. The largest subunit of Pol II (referred to as RPB1 or RPB205) is a 1,840 amino acid protein that contains one C2H2-type zinc finger and a C-terminal domain comprised of several heptapeptide repeats. Although Pol II function requires the cooperation of all twelve subunits, the largest subunit conveys Pol II catalytic activity and, together with the second largest subunit, forms the active center of the Pol II enzyme. Additionally, the large subunit participates in forming the DNA-binding domain of Pol II, a groove that is necessary for transcription of the DNA template. Without proper function of the large subunit, mRNA synthesis and subsequent transcription elongation cannot occur.
Description: RNA polymerase II (Pol II) is an enzyme that is composed of 12 subunits and is responsible for the transcription of protein-coding genes. Transcription initiation requires Pol II-mediated recruitment of transcription machinery to a target promoter, thereby allowing transcription to begin. The largest subunit of Pol II (referred to as RPB1 or RPB205) is a 1,840 amino acid protein that contains one C2H2-type zinc finger and a C-terminal domain comprised of several heptapeptide repeats. Although Pol II function requires the cooperation of all twelve subunits, the largest subunit conveys Pol II catalytic activity and, together with the second largest subunit, forms the active center of the Pol II enzyme. Additionally, the large subunit participates in forming the DNA-binding domain of Pol II, a groove that is necessary for transcription of the DNA template. Without proper function of the large subunit, mRNA synthesis and subsequent transcription elongation cannot occur.
Description: RNA polymerase II (Pol II) is an enzyme that is composed of 12 subunits and is responsible for the transcription of protein-coding genes. Transcription initiation requires Pol II-mediated recruitment of transcription machinery to a target promoter, thereby allowing transcription to begin. The largest subunit of Pol II (referred to as RPB1 or RPB205) is a 1,840 amino acid protein that contains one C2H2-type zinc finger and a C-terminal domain comprised of several heptapeptide repeats. Although Pol II function requires the cooperation of all twelve subunits, the largest subunit conveys Pol II catalytic activity and, together with the second largest subunit, forms the active center of the Pol II enzyme. Additionally, the large subunit participates in forming the DNA-binding domain of Pol II, a groove that is necessary for transcription of the DNA template. Without proper function of the large subunit, mRNA synthesis and subsequent transcription elongation cannot occur.
Description: TAF10 Human Recombinant produced in E.coli is a single, non-glycosylated polypeptide chain containing 158 amino acids (84-218) and having a molecular mass of 16.9kDa.;TAF10 is fused to a 23 amino acid His-tag at N-terminus & purified by proprietary chromatographic techniques.
Description: This product includes all the reagents in E. coli RNA Polymerase Assay Kit (Catalog number RPA100K) plus the enzyme, 33 ul of 100 x E. coli RNAP. It is for 100 assays of E. coli RNA polymerase reactions in a 384-well assay format.
Elongation Factor RNA Polymerase II (ELL) Blocking Peptide
Description: Mouse polyclonal to RNA polymerase II CTD repeat YSPTSPS
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The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was an extended variation between the sequences of the same individual as between the sequences of different individuals. Interpanding variability was significantly lower in people infected with a common source. A high proportion of amino acid substitutions in hypervariable regions alter the number and positions of potential glycosylation sites n-linked.
