Principle and Troubleshooting

The technical benefit of multiplex PCR over conventional PCR is that in multiplex PCR more than one target sequence can be simultaneously amplified by including more than one pair of primers in the PCR reaction. The optimization of multiplex PCRs may result in some difficulties and the main one is the preferential amplification of certain specific targets. The presence of more than one primer pair in the PCR reaction increases the chance of obtaining nondesirable amplification products, primarily because of the formation of primer dimers. Nonspecific products may be amplified more efficiently than the desired target producing impaired rates of annealing and extension. Trial-and-error approach may have to be used when testing several primer pairs, because there are no means to predict the performance features of a putative primer pair even among those that satisfy the general parameters of primer design. Special attention to the homology of primers with their respective target sequences, their GC content, and concentration, and obviously avoiding inter- and intraprimer homology have to be considered. All the primer pairs in a multiplex PCR should enable similar amplification efficiencies for their respective target. Polymerase chain reaction selection is defined as a mechanism that favors the amplification of certain templates due to the properties of the target or the target's flanking sequences. The choice of primers has been shown to be crucial to avoid PCR selection. A primer length of 18 bp or larger and a GC content around 40% and with identical or very close optimum annealing temperatures should not display any significant homology either internally or to one another could be assayed. Optimization of PCR components such as PCR buffer constituents, dNTPs, enzyme concentrations, and PCR additives, such as bovine serum albumin, dimethyl sulfo-xide, glycerol, or betaine, has been reported to be useful in multiplex PCRs. These additives may act to avoid the stalling of DNA polymerization, which may occur through the formation of secondary structures within regions of template DNA during the extension process. Such cosolvents may also act as destabilizing agents, reducing the melting temperature of GC-rich sequences, or as osmoprotectants, increasing the resistance of the polymerase to denaturation. The primer pairs must be inclusive for as many strains of the target microbe as possible, and depending on the amplicon detection method, their targets are easily resolvable. The latter may be achieved by using primer pairs that result in PCR products that can be separated and clearly visualized using gel electrophoresis or hybridization probes with maximum specificity.

Many multiplex PCRs developed before utilized a nested strategy. Nesting significantly increases the sensitivity of the assay but also increases the chance of false-positive results due to contamination and may also complicate automation.

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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