Laser Induced Fluorescence LIF

Laser-induced fluorescence is a standard detection method in a majority of the automated sequencers. This method is extremely sensitive, allows automation of base calling, avoids using radioactive materials, and promotes a throughput increase by using several spectrally distinct labels simultaneously. Laser-induced fluorescence is based on the excitation of a dye label attached to a DNA molecule by laser light. Resulting fluorescence is collected and converted to an electrical signal by a photosensitive element [photomultiplier tube (PMT), photodiode, or chargecoupled device (CCD) element]. The output signal (trace) represents a series of peaks each corresponding to a DNA chain of a certain size. One trace provides information about one of the four bases (A, C, G, or T). Therefore, in order to obtain information about a complete sequence, four traces are required. This is achieved either by using four physically separated lanes (or capillaries) or by running four differently labeled samples in a single lane.

If a single laser is used in the sequencer, different parts of the gel or different capillaries are illuminated through a scanning objective lens. Alternatively, a sheath cell[5] can be used with simultaneous illumination of several DNA traces. Some DNA sequencers contain several lasers either with different wavelengths improving excitation[6] or with the same wavelength providing excitation of multiple samples without using moving parts.[7]

Collected fluorescence is converted into digital form and processed by a computer. Software packages used in different sequencers vary in their functionality and efficiency, but all of them involve the following common steps: noise reduction, base line subtraction, color crosstalk compensation, trace alignment, and peak recognition. A typical example of the resulting base calling is shown in Fig. 1.

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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