CONTRIBUTION OF ERRORS INDUCED BY THE TAQ POLYMERASE TO ESTIMATE THE DIVERSITY OF RNA VIRUS.
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CONTRIBUTION OF ERRORS INDUCED BY THE TAQ POLYMERASE TO ESTIMATE THE DIVERSITY OF RNA VIRUS.
The genetic diversity of a vesicular stomatitis virus population was analyzed by RT-PCR, cloning and sequencing of about 500 nucleotide regions of the virus genome. PCR amplifications were performed in parallel experiments with DNA Taq and PFU polymerases and significant differences were observed. Between 10 and 22 mutations were detected when virus populations were analyzed by TAQ amplification (20 clones of each region), while the amplification of the same samples with PFU revealed between 0 and 5 mutations. PCR loyalty tests, carried out under the same PCR conditions as those used in the population analysis, have shown that the Taq error rate estimation of 0.27 x 10 (-4) Enporations by PB by Cycle was included in the range estimated elsewhere of the amplification of the recombinant PCR plasmids (0.27-0.85 x 10 (-4) Errors by BP per cycle) or functional tests (0.2-2 x 10 ( -4) Errors by PB per cycle).
The TAQ error rate has been found to be 9.3 times higher than the PFU error rate with DNA as a model, and about 10 times higher with the CDNCs obtained by inverse transcription of models. Viral RNA from natural populations. In this study, we discuss (i) the implications of TAQ errors on the analysis of genetic variability, based on both frequency and nature (synonymous replacement) of the substitutions observed and (ii) the size of the Sample required to evaluate genetics variability in a virus population generated by a single infection. In this project, a real-time polymerase chain reaction (PCR) was used to study the mechanism of PCR inhibition by examining the effect of the amplicon length, the melting temperature and of the sequence.
First specially designed with three different amplicon lengths and three different melting temperatures have been used to target a single homozygous allele in the Humert01 locus. The effect on the efficiency of the amplification of each pair of primers has been determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that various inhibition mechanisms can occur during the PCR process based on the type of co-extract inhibitor. These include TAQ inhibition, the binding of DNA templates and the effects on the efficiency of the reaction.
PCR-resistant PCR-resistant Taq DNA mutants allow for amplification of total blood DNA and gross soil samples.
Powerful PCR inhibitors in blood and soil samples can cause false negative results of PCR-based clinical and lawy tests. We show that the effect of these inhibitors is mainly on the TAQ DNA polymerase, because the mutational modification of the polymerase can overcome the inhibition insofar as no purification of the DNA is now required. N-terminal removal (Klentaq1) is an inhibition of 10 to 100 times resistant to total blood relative to the Taq Wild Type TAQ (W.T.) Taq, which is strongly inhibited by 0.1 to 1% blood. Other mutations at Codon 708, both in Klentaq 1 and Taq, give increased resistance to various PCR reaction inhibitors, including total blood, plasma, hemoglobin, lactoferrin, IgG. Serum, soil extracts and humic acid, as well as high concentrations of inserted dyes.
The blood PCR inhibitors can mainly reduce the speed of extension of the W.T DNA Taq polymerase with respect to mutant enzymes. Human genomic targets to a human copy are easily amplified from total blood or raw soil extract, without pretreatment to purify the template DNA, and the permitted increase in the dye concentration overcomes the background of Fluorescence and quenching in real-time blood PCR.
CONTRIBUTION OF ERRORS INDUCED BY THE TAQ POLYMERASE TO ESTIMATE THE DIVERSITY OF RNA VIRUS.
DNA removal contaminant in the reaction reagents of the polymerase chain: implications for a general approach to the detection of unutaneous pathogens.
The analysis based on comparisons of RNA 16S sequences provides a quick and reliable approach to identifying human pathogens. By leading the primers of oligonucleotides to the sequences preserved throughout the kingdom of ebacterial, the bacterial ribosomal DNA sequences of substantially any member of the kingdom of the ebacteria can be amplified by the reaction of the chain of the polymerase and subsequently analyzed by the determination of the sequence. Indeed, automated wide range amplification systems, sequencing and data analysis are now feasible and can be founded on the next generation of automated microbial identification systems.
However, the identification of the pathogens by this strategy is hampered by the frequent contamination of the reagents used for the amplification reaction, in particular the Taq polymerase, with an exogenous bacterial DNA. Here, we describe detailed surveys of the use of 8-methoxypsorane UV light and the long waves to remove the contaminant DNA in the reaction reagents of the polymerase chain. The clinical utility of the developed procedure has been demonstrated in a case of pacifacillary osteomyelitis for which no specific bacterial agent had been cultivated. We have developed and characterized a test for the interactive compounds of G-quadruplex that use the fact that G-rich DNA models have barriers to the synthesis of DNA by DNA polymerases.
Recombinant Measles Virus MV Large Polymerase 58-149 a. a Protein, Untagged, E.coli-100ug
Description: Recombinant MeV Large Polymerase containing the large polymerase immunodominant regions, 58-149 amino acids was expressed in E. coli and purified by proprietary chromatographic technique.
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.
Use of Taq DNA polymerase and G-quadruplex 2, 6-diamidoantoanthraquinone bonding BSU-1051, we find that the BSU-1051 leads to improved arrest of the synthesis of the DNA in the presence of K + by stabilizing an intramolecular structure G-quadruplex formed by four repetitions of TTGGGG or TTAGGG in the model pane. The data provides additional evidence that BSU-1051 modules the telomerase activity by stabilizing G-quadruple telomer DNA and point to a polymerase stop test as a sensitive method for screening for interactive agents of G- Quadruplex with potential clinical utility.
