Synthesis of Competitors

The construction of competitor templates, their exact quantification, and their storage represent the crucial aspects of this technique. Competitors, to act as a functionally appropriate internal standard, must behave during the assay as closely as possible to the template. The efficiency of the enzymatic reactions (reverse transcriptase and PCR) on the two species must be comparable.[5,6] For this reason, the competitor must have comparable dimensions and the same primer recognition sites as the target. However, for the assay to be practical, the separation and the measurement of the two species should be performed in the simplest way possible. Different strategies have been described for the construction of the competitor for c-PCR. Some competitors have been obtained by the incorporation of a new restriction site in the native sequence,[7] and after PCR, the competitor is digested with the relevant restriction enzyme and the electrophoretic bands are then resolved and measured. The main disadvantage of this approach is the unpredictable efficiency of enzyme digestion, which can strongly influence the results of the assay. A variant of this technique is based on the insertion of a small modification in nucleotide sequence of the target (short sequence modification or single-base mutation).[8] After PCR, the competitor can be identified by differential hybridization with specific probes or by electrophoretic techniques [denaturating gradient gel electrophoresis (DGGE) or single-strand conformation polymorphism (SSCP1)]. This approach is quite complex and requires time-consuming and cumbersome post-PCR procedures. Furthermore, the

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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