and also quite thin, and the thermal ramps are relatively shallow, reasonable spatial thermal homogeneity is achieved, at least to within 0.1 °C as determined by a reference platinum resistivity thermometer. Alternately, an electrophoresis box with a fast impellor, a rack for the gels, and a glass serpent taking the input from a programmable, recirculating water bath can be used. The latter are commercially available and the remainder is simple to build. At 10 MADGE gels per tank (96-well or 192-well) and 1-4 hr run time (faster if powerful cooling is available to counteract amperometric heating), it is feasible for one worker to optimize a number of amplicon assays in parallel, or to run several thousand scans (either of thousands of subjects for one amplicon or of several amplicons of matched melting characteristics for a few hundred subjects) per day. Assay optimization tends to be the limiting factor.
The reconfiguration of the denaturing gradient to have temperature as the dependent variable and time as the independent (controlled) variable opens a range of significant advantages:
1. It creates the option for high density arraying of wells and tracks (e.g., MADGE arrays), hence enabling high throughput of analyses.
2. Programmability of the gradient, which can be arbitrary and uses temperature rather than chemicals, is achievable easily in time. Arbitrary ramps (rather than simple linear gradients) would be very difficult in space. Gradients as an integral feature of the gel demand special gels. Programmability gives flexibility and convenience.
3. Potential for arbitrary ramps enables very short track lengths, and short run time (e.g., 1 hr) further enables high track density and hence throughput.
4. The use of ordered arrays (e.g., microplate format of MADGE) greatly simplifies data handling.
5. Arbitrary programming enables arbitrary resolution, thus reducing the analysis to simple pattern recognition rather than detailed relative mobility measurements. Pattern recognition evades the need for closely juxtaposed tracks or for internal markers. By contrast, the resolution of single-stranded conformation polymorphism (SSCP) is often small, cannot be programmed, and hence cannot be arbitrarily great. Thus advantages 1-4 cannot be realized with SSCP.
We have applied this system to a number of human genes both for studies of large case collections and for epidemiological research in population samples (BRCA1 in breast cancer risk; LDLR in familial hypercholesterolemia; MC4R-population research in obesity; APOE). Other assays relevant to the diagnostic laboratory are under development.
Identification of unknown mutations is an important aspect in genetic diagnostics. For mutations not known in advance, the choice at present is between direct sequencing, de novo scanning techniques to target sequencing more efficiently to appropriate regions, and ''chip'' technologies equivalent with direct resequencing by multiple parallel oligonucleotide hybridization. For the identification of rare variants, important criteria are the degree of parallelism of samples possible, the number of bases analyzed per ''run,'' and the total number of bases an individual can scan per day. For heterozygote sequencing in humans, the difficulties of accurate base calling (essential for every base to avoid excessive numbers of false positive calls) may restrict sequencing as a first-line approach. Even as sequencing improves, reagent requirements and need for high-resolution elec-trophoresis introduce significant cost issues. Chip technologies, while promising, have low capability for sample parallelism; a chip is expensive ($5000-10,000) for any selected sequence and also requires an optimization process; and the approach will demand high capital expenditure on chip-reading hardware. Scanning technologies are much used, but intense activity to develop improved approaches reflects the importance and shortfalls in this field. Such technologies generally rely on elec-trophoresis and give some information about the mutation from the resultant band pattern. MeltMADGE enters the field alongside SSCP, denaturing gradient gel electrophoresis (DGGE), and denaturing HPLC. Sensitivity to all base changes has been a concern with SSCP. Denaturing gradient gel electrophoresis, although used in diagnostics laboratories on account of good sensitivity to most if not all base changes, is laborious and has low throughput. Denaturing HPLC has recently become prevalent but incurs high capital costs and high consumables costs, both tenfold greater than for the other techniques. In effect, meltMADGE should be considered as a convenient, high-throughput format of DGGE. Our comparisons with other techniques (to be published) show good sensitivity to most base changes, given appropriately planned and optimized assays.
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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.