Well MADGE and 768Well MADGE

384-well microplates have become an established new standard in higher-throughput genomics research laboratories for clone operations (arrayed libraries, gridding, spotting, storage, etc.) and for PCR. The difficulties in thin-walled plastics manufactured for the latter have been solved. 384-well microplates have wells in a 16 x 24, 4.5-mm pitch array. A diagonal turn of this array would result in MADGE wells and tracks too narrow (< 1 mm) for manual access, but we have devised a 384-well MADGE format which combines four 96-well arrays in a linear (rather than tetradic) overlay. The 384-format locates the long axis of the array at 11.3° relative to the direction of electrophoresis and respectively give track lengths of 9.98 and 4.24 mm and track width of 1.5 mm. The 1.5 x 1.5 x 1.5-mm wells can be accessed by human hand. However, the array is dense and confusing for 8- or 12-channel loading, but 96-pin passive or air displacement transfer is not a problem for human hand and eye. We have also derived a 768-well format from 384[9] in the same way that the 192-well format was derived from the original 96-well format (see above). Protocols for setting up 768-well gels are identical with those for 384-well gels. Unlike 96- and 192-well protocols, the 384-well protocol depends on the use of oil-free PCR, 96-pin manual transfers from 384-well PCR plate to gel, and use of dry rather than submerged gels to enable the use of passive pin transfers. However, this approach is also considerably more convenient. Combined with direct electrode contact in ''dry'' clamshell electrophoresis boxes which plug directly into contacts in a powered stacking frame and using 5-10 min electrophoresis times, very much higher throughputs can be achieved than with 96-well submersible systems. Components of this system are shown in Fig. 1C and D and a typical result from a 384-well gel is shown in Fig. 2. The present approach to machining of the gel formers is near its limit because the space between ''teeth'' is small, but other arrangements of teeth, or other approaches to gel former production, may enable yet higher densities. However, it is likely that this would force the transition from economy of startup and versatility of

Fig. 2 384-well MADGE gel used for dual-reaction ARMS assay. On the main gel image the red rectangle indicates reactions a and b for sample A1, array 1, whereas the blue rectangle indicates reactions a and b for sample A1, array 2. The enlargement shows the three possible genotype calls as pairs of lanes with either two bands in each heterozygote or two bands in one and one in the other (for each category of homozygote). (View this art in color at www.dekker.com.)

Fig. 2 384-well MADGE gel used for dual-reaction ARMS assay. On the main gel image the red rectangle indicates reactions a and b for sample A1, array 1, whereas the blue rectangle indicates reactions a and b for sample A1, array 2. The enlargement shows the three possible genotype calls as pairs of lanes with either two bands in each heterozygote or two bands in one and one in the other (for each category of homozygote). (View this art in color at www.dekker.com.)

the human operator to expensive hardware configured around a core mini-microscale format, and at this level, complete departure from industry-standard microplate format may be appropriate.

High-Resolution 96-Well MADGE (StretchMADGE) for Sizing Microsatellite Alleles

The following modifications enable resolution of bands with small (e.g., 1.5-5%) mobility differences (Fig. 3A):

• Increased track length on 2 x 6 diagonal instead of 2 x 4 diagonal.

• Introduction of internal molecular weight markers in every track.

• Sample dilution to avoid salt artefacts in the electrophoresis.

• Use of better resolving acrylamide derivatives such as duracryl.

• Thermostatic control of the electrophoresis.

This has enabled calling of minisatellite[10] and tetranucleotide and trinucleotide repeat multiallelic micro-

Fig. 3 StretchMADGE and meltMADGE. (A) StretchMADGE resolution of homoduplexes and heteroduplexes of a tetranucleotide microsatellite amplicon with five main alleles. Except in homozygotes, two resolving homoduplexes and two resolving or corunning heteroduplexes are observed. Homo-duplexes are sized relative to flanking size markers (arrows) introduced into every track. Amplicon size is ~ 260 bp, so one repeat size difference corresponds with approximately 1.5% band mobility difference. (B) MeltMADGE scanning of an amplicon for identification of unknown mutations. As explained in the text, heterozygotes for a mutation show a set of bands (tracks marked by arrows) instead of a single band, representing differential melting and hence differential mobilities during thermally ramped electrophoresis.

Fig. 3 StretchMADGE and meltMADGE. (A) StretchMADGE resolution of homoduplexes and heteroduplexes of a tetranucleotide microsatellite amplicon with five main alleles. Except in homozygotes, two resolving homoduplexes and two resolving or corunning heteroduplexes are observed. Homo-duplexes are sized relative to flanking size markers (arrows) introduced into every track. Amplicon size is ~ 260 bp, so one repeat size difference corresponds with approximately 1.5% band mobility difference. (B) MeltMADGE scanning of an amplicon for identification of unknown mutations. As explained in the text, heterozygotes for a mutation show a set of bands (tracks marked by arrows) instead of a single band, representing differential melting and hence differential mobilities during thermally ramped electrophoresis.

satellite polymorphisms.1-11-1 We have been interested in using such sites within genes as linkage disequilibrium markers in association studies and wider utility can be anticipated. In our system, microsatellite amplicons are electrophoresed double stranded and there is the possibility that not only length polymorphic information but also internal sequence variation will be accessible by examining heteroduplex mobility.

Temporal Thermal Ramp MADGE Electrophoresis (MeltMADGE) for Identification of Unknown Mutations

As MADGE involves a complex spatial configuration of sample wells and tracks, electrophoresis methods involving spatial gradients would be difficult to establish. Denaturing gradient gel electrophoresis,1-12-1 which is used for identification of unknown mutations, involves a spatial gradient of denaturant. Polymerase chain reaction products heterozygous for a base change possess four moieties resolvable as separate bands. Consider a G to A transition. The homoduplex with a G-C base pair will migrate furthest in the gel because it will be the last moiety to denature with consequent reduction of migration velocity. The homoduplex with an A-T base pair will migrate almost as far. The heteroduplex with the relatively stable G-T mismatch will show a band of lower mobility. The heteroduplex containing the relatively unstable A-C mismatch will appear as the band nearest to the well. A thermal ramp in time can achieve a similar effect to a denaturing gradient in space and has the advantage of spatial homogeneity and hence applicability to the MADGE (or other array) formats. This forms the basis of our meltMADGE invention[13] and an early proof of principle example is shown in Fig. 3B.

We have previously described (for other use than meltMADGE) a purpose-built programmable real-time thermoregulated electrophoresis apparatus.[14] In brief, a 10x 10x 15-cm electrophoresis tank contains buffer which is continuously mixed to ensure spatial thermal homogeneity, and the temperature is varied real-time by programmable software controlling heating and cooling systems and receiving feedback from temperature sensors in the tank. Overall, this is little more complex than a PCR machine, although for PCR low thermal mass for rapid temperature shifts is the objective, whereas for melt-MADGE, the aim is high thermal mass for stability of small temperature shifts. The initial prototype accommodates 10-12 horizontal MADGE gels spaced in a carrier stack. Each 2-mm-thick gel is adherent to and supported by a 2-mm glass plate and is also covered by 2-mm glass, with spacing between each glass/gel/glass sandwich for buffer circulation. As buffer and hence thermal circulation is vigorous and the glass is both a good thermal conductor

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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