Technical Description

The DGGE method is based on the electrophoretic mobility of a double-strand DNA molecule through linearly increasing concentrations of a denaturing agent (urea and formamide) on a polyacrylamide gel with ethidium bromide staining. As the DNA fragment proceeds through the gradient gel, it will reach a position where the melting temperature (Tm) of its lowest melting domain equals the denaturing agent concentration, resulting in denaturation and consequent marked retardation of the DNA fragment mobility. As the Tm of a melting domain is dependent on its nucleotide sequence, even DNA fragments differing by a single nucleotide in their lowest domain will suffer branching and consequent retardation of their mobility at different positions along the DGGE gel, allowing DNA fragment separation.[2] The choice of the gel denaturant range is based upon the Tm of the fragment to be analyzed, and the electrophoretic runs can be performed at a constant temperature (58°C) that exceeds the Tm of an A-T-rich DNA fragment in the absence of denaturing agents.

Denaturing gradient gel electrophoresis cannot resolve fragments differing by nucleotide changes in the highest melting domain because of complete strand dissociation. This problem is overcome by introducing a GC clamp tail as short as 40 bp to serve as a high Tm domain and prevent complete dissociation of the DNA fragment. The GC clamp tail introduction increases the DGGE mutation detection percentage to close to 100%.[1] It is also possible to split DNA fragments into two segments to allow efficient mutation detection. The melting behavior of a DNA fragment can be simulated by computer software analysis.[3]

The DGGE method is conceptually similar to hetero-duplex analysis.[4,5] The PCR-DGGE combination is extremely efficient when applied to heterozygous nucle-otide variants because of continuous denaturation and reannealing of single-strand molecules during PCR, allowing for the formation of heteroduplex and homo-duplex molecules.[6] The presence of a single nucleotide change within heteroduplexes decreases their melting domain temperature allowing separation from the homo-duplexes and easy visual detection of the mutants (Fig. 1). If working with an X-linked disease it is necessary to mix male mutated DNA with a normal male DNA during PCR to ensure heteroduplex formation. To reduce workload, DNA fragments can be amplified in multiplex combinations. A further sequencing of the altered DNA fragment can determine the exact molecular alteration.

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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