This ready-to-use nucleotide mix is a premixed solution of the sodium salts of dATP, dGTP, and dCTP, each at a concentration of 10 mM, and dUTP at a concentration of 30 mM in water. This mix is optimized for use in all types of amplification reactions: PCR
RT-PCR
Prevention of carryover contamination
To allow decontamination of PCR or RT-PCR, dUTP in place of dTTP is incorporated into the PCR product. Subsequent reactions may then be treated with Uracil-DNA Glycosylase (UNG). Avoiding the need of re-opening the reaction vial, the vials are incubated at +20 °C, resulting in the degradation of potentially contaminating uracil-containing amplification products. During this step, template DNA and RNA remain unaffected, since normal DNA does not contain uracil, and RNA does not serve as a substrate for UNG. Before starting the actual thermocycling program, UNG is inactivated by incubation at +95 °C. Uracil -DNA Glycosylase, heat-labile is particularily useful, as it is fully inactivated already after incubation at +95 °C for 2 minutes. The natural enzyme from E. coli requires incubating the reaction mixture for 10 minutes at +95 °C. The shorter heat treatment substantially reduces the risk for loosing the template nucleic acid, which typically is present at low concentrations only. This is of particular importance, when performing RT-PCR. We therefore recommend the use of Uracil-DNA Glycosylase. It has been used in LightCycler PCR assay to identify B. pertussis and B.parapertussis in nasopharyngeal swabs. It has also been used in quantitative PCR (qPCR) assay.
一般的な説明
PCR Nucleotide MixPLUS is a clear, colorless solution of the sodium salts of dATP, dCTP, dGTP, each at a concentration of 10 mM, and dUTP at a concentration of 30 mM in Water, PCR Grade. This nucleotide mixture can be added directly to polymerase chain reactions. The incorporation of dUTP in place of dTTP allows the degradation of contaminating PCR products from former reactions with Uracil-DNA Glycosylase (UNG) to prevent carryover contamination from previous amplifications.
