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.
POLR2B, ID (POLR2B, DNA-directed RNA polymerase II subunit RPB2, DNA-directed RNA polymerase II 140kD polypeptide, DNA-directed RNA polymerase II subunit B, RNA polymerase II subunit 2, RNA polymerase II subunit B2)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) APC
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) APC
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (Biotin)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (Biotin)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (AP)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (AP)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (FITC)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (FITC)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (HRP)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (HRP)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (PE)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (PE)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 490)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 490)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 550)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 550)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 405)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 405)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 650)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 650)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 750)
POLR2A (DNA-directed RNA Polymerase II Subunit RPB1, RNA Polymerase II Subunit B1, DNA-directed RNA Polymerase II Subunit A, DNA-directed RNA Polymerase III Largest Subunit, RNA-directed RNA Polymerase II Subunit RPB1, POLR2, MGC75453) (MaxLight 750)
Description: RNA polymerase II-IN-1 (compound 19iv) is a amatoxin, inhibiting RNA polymerase II (Pol II) with an IC50 value of 36.66 nM. RNA polymerase II-IN-1 has higher cytotoxicity against cancer cells and less toxic in normal cells than α-Amanitin[1].
Description: RNA polymerase II-IN-2 (compound 20iii) is a potent RNA polymerase II (Pol II) inhibitor with Ki value of 9.5 nM. RNA polymerase II-IN-2 has cytotoxicity against cancer cells, and exhibits 2 and 5 fold toxicity than α-amanitin against CHO and HEK293[1].
POLR2G, CT (POLR2G, RPB7, DNA-directed RNA polymerase II subunit RPB7, DNA-directed RNA polymerase II subunit G, RNA polymerase II 19kD subunit)
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.
