Despite their attraction, there are several problems associated with the use of nonspecific dyes. In particular, there is a broadening of the melting transition and Tm compression among genotypes, which can lead to ambiguous results. Furthermore, dsDNA-specific dyes may redistribute during melting causing the release of dye from low-melting heteroduplexes and redistribution to higher melting heteroduplexes. The use of labeled primers for melting analysis avoids these problems while retaining the advantage of not having to use specific probes for each assay. The use of one fluorophore-labeled and one unlabeled primer enables melting profiles of the amplicon to be obtained immediately after completion of the PCR reaction and results in distinct melting curve shapes for different alleles, including those that differ by only a single base.
Another method makes use of the principle of fluorescence resonance energy transfer (FRET). This is a distance-dependent interaction between the excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule at distances up to 70-100 A without emission of a photon. As a result, the emission of the reporter fluorophore is quenched. In this approach, one of the amplification primers has a reporter and a quencher moiety attached to a hairpin structure at its 5' end (Fig. 4A). When in solution, fluorescence emission from the reporter is quenched. A fluorescent signal is generated only when the labeled oligonucleotides are incorporated into the ds amplification product. Labeled primer synthesis has been simplified recently by using the same type of hairpin primer, but only a single reporter fluorophore whose fluorescence emission is self-quenched based on sequence context. These primers, also known as LUX™, quench when free in solution, fluoresce weakly when denatured, and emit light strongly when incorporated into DNA (Fig. 4B). Differential fluorescence labeling of primers allows the use of allele-specific PCR in a single tube.
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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.