Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.
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Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.
A set of universal oligonucleotide primers for the retained regions of the eubactterial gene 16S arrnes has been designed for use with the 7700 Real-Time PCR Applied Biosystems (Taqman). During the development of this PCR, problems have been noted with the use of this gene as an amplification target. The contamination of reagents with bacterial DNA was a major problem exacerbated by the very sensitive nature of real-time PCR chemistry. This has been aggravated by the use of a small amplic of about 100 bases, as it is necessary with Taqman chemistry. In an attempt to overcome this problem, several methodologies have been applied. Some treatments were more efficient than others to eliminate contaminant DNA; However, to achieve this, there was a decrease in sensitivity.
With UV irradiation, a 4-log reduction of PCR sensitivity, with UV-facing 8-methoxypsorore activity, there was a 5 to 7 log discount, and with DNASE alone and in combination with a restriction digestion, There was a 1.66- reduction of the newspaper. Treatment of restriction endonuclease alone and together did not reduce the level of contaminant DNA. Without the development of Taq DNA polymerase ultrapide, ultrapular reagents and guaranteed plastic to be DNA free, the implementation of a PCR for the detection of the 16S Eubacterial 16S RNA with the Taqman system will continue to continue to be problematic. For three Taq polymerases from different commercial sources, the MICs were 0.08 to 0.64 micrograms of standard humic acids per ml and 0.24 to 0.48 micrograms of the moist acids coextraits per ml. The addition of the T4 Gene 32 protein increased the microphone for a 5.12 micrograms / ml taq.
Humic acids have decreased non-translative detection in DNA-DNA shoulder hybridizations at amounts of 0.1 micrograms and inhibited transformation of Escherichia coli coli HB101 with a plasmid with wide range of hosts, PUN1, at concentrations of 100 micrograms / ml. The purification of the gross DNA with ion exchange chromatography resulted in a 97% removal of the initially coextraits humic acids.
Quantity of triphosphates of cellular deoxynucleoside.
The eukaryotic cells contain a delicate scale of minus quantities of the four triphosphates of deoxyribonucleoside (DNTP), sufficient only for a few minutes of DNA replication. A deficiency and a single DNTP surplus can increase mutation rates, defective DNA repair or a depletion of mitochondrial DNA. DNTPs are generally quantified by an enzymatic test in which the incorporation of radioactive DATP (or radioactive DTTP into the DATP test) in synthetic oligonucleotides specific by a polymerase DNA is proportional to the concentration of the unknown DNTP. We find that the Klenow DNA polymerase commonly used can substitute the corresponding ribonucleotide for the unknown DNTP leading in some cases to a large overestimate of DNTPs.
We now describe the dosing conditions for each DNTP that avoid the constitution of ribonucleotides. For DTTP and DATP analyzes, it is sufficient to minimize the concentrations of the Klenow enzyme and marked DATP (or DTTP); For DCTP and DGTP, we had to replace Klenow’s enzyme with the polymerase of Taq DNA or thermal sequenease. We suggest that in some previous reports, the constitution of ribonucleotide can have caused too high values for DGTP and DCTP. A new direct detection method of PCR amplified DNA in a closed system is described.
The method is based on the incorporation of primers labeled by energy transfer into the amplification product. PCR primers contain hairpin structures on their 5 years with fragments of donors and acceptors near the hairdressing rod. The primers are designed so that a fluorescent signal is generated only when the primers are incorporated into an amplification product.
Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.
Effects of amplification facilitators on diagnostic PCR in the presence of blood, fecal materials and meat.
The full potential of diagnostic PCR is limited, in part, through the presence of inhibitors in complex biological samples that reduce the efficiency of amplification. Therefore, various pre-PCR treatments are used to reduce the effects of PCR inhibitors. The objective of this study was to study the effects of 16 amplification facilitators to improve the amplification of DNA in the presence of blood, fecal or meat. Different concentrations of amplifying adapters and inhibitory samples were added to PCR mixtures containing RTTH or TAQ DNA polymerase. The addition of bovine serum albumin of 0.6% (WT / vol) on reaction mixtures containing DNA Taq polymerase reduced the inhibitory effect of blood and authorized the amplification of the DNA in the presence 2% instead of 0.2% blood (flight / vol). In addition, the addition of bovine serum albumin (BSA) with reaction mixtures containing faecal materials or meat has improved the amplification capabilities of the two polymerases.
The Taq DNA The polymerase was able to amplify the DNA in the presence of 4% instead of 0.4% (theft / vol) of faeces and 4% instead of 0.2% (flight / vol) , and RTTH was able to amplify the DNA in the presence of 4% instead of 0.4% (theft / vol) of feces and 20% instead of 2% meat (flight / vol). The T4 Gene T4 (GP32) protein (GP32) of the DNA bonded binding has presented an effect of relief similar to that of the BSA, except when it has been added to PCR mixtures of the meat containing and from The TAQ DNA Polymerase containing faeces. The effects of beetaine relief and a cocktail of proteinase inhibitors were more specific samples.
The addition of 11.7% (WT / Vol) betaine has allowed the Taq DNA polymerase to amplify the DNA in the presence of 2% blood (theft / vol), while the addition of inhibitors Proteinase has led to an amplification of the DNA by the two polymerases in the presence of 4% (theft / vol) features. When different combinations of betaine, BSA, GP32 and proteinase inhibitors have been tested, no synergistic or additive effects have been observed. The effects of the facilitators on the synthesis of DNA in real time instead of conventional PCR have also been studied.
