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Allele 1-Specific PCR Product

Allele 2-Specific PCR Product

Single Molecule Fluorescence and

Genotype Assignment

Single Molecule Fluorescence and

Genotype Assignment

Fig. 1 The principle of biallelic SNP scoring with GALIOS™. (View this art in color at www.dekker.com.)

GALIOSTM reaction, these tailed PCR products serve as a template for the universal labeling primers, finally resulting in allele-specific accumulation of fluorescent amplification products.

After the GALIOSTM amplification, reaction mixes are analyzed directly, without any post-PCR processing or physical separation steps, by single molecule fluorescence (i.e., fluorescence correlation spectroscopy or FCS).[7-10]

Whereas traditional macroscopic fluorescence techniques monitor the average fluorescence output from the entire ensemble of emitting fluorophores in a given reaction, single molecule fluorescence employs confocal optics with a very tiny illumination volume of approximately 1 fL (10_ 15 L). Only fluorescence emitted within this confocal volume is observed and traced in a time-resolved manner. The resulting fluorescence raw data are amenable to different types of fluctuation analysis including FCS, which statistically samples the fluorescence fluctuations emitted from single molecules in discrete time intervals, with a temporal resolution down to microseconds and below. This process is termed autocorrelation and provides information on multiple fluorescence parameters including the average number, the molecular brightness, the triplet fraction, and the transla-tional diffusion time of fluorescent molecules within the confocal volume. By fitting the autocorrelated fluorescence signal to a two-component model, the relative amounts of two species of fluorescent molecules differing in their translational diffusion time (e.g., the amounts of free universal primer and fluorescent PCR product in GALIOSTM reactions) are determined.

By combining confocal optics and autocorrelation, FCS virtually eliminates problems associated with conventional macroscopic fluorescence techniques, such as probe adsorption, inner filter effects, photobleaching of fluorophores, and particularly the decrease of signal with decreasing reaction volumes (i.e., with miniaturization). Thus, FCS provides a high signal-to-noise ratio (SNR) and a maximum sensitivity that is essentially independent of the assay volume. This allows ultimate assay miniaturization down to 1 pL or below sustaining a consistent signal quality—a favorable feature for economical high-throughput SNP genotyping. In addition, the obtained multiple fluorescence parameters serve as intrinsic quality controls within each reaction.

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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