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CastAway Precast Gels for Rapid Automated DNA Sequence Analysis

assure optimal signal-to-noise ratios for a given gel configuration (Figure 2).

Figure 2

DNA reaction products are heat denatured and loaded on the CastAway gel using a 48- or 64-well sharktooth comb. To separate DNA fragments produced in a sequencing reaction, electrophoresis is carried out at a constant 25 W (ca. 50 V/cm and 18 to 22 mA) (Figure 3). To obtain banding patterns of short tandem repeat polymorphisms (STRPs), fluorescently labeled PCR products are separated using electric field conditions of 1000 V, 45 mA, and 45 W (Figure 4). During electrophoresis, the precast gel is maintained at 45C by a thermostatically controlled heater plate that is in contact with the rear glass plate. After electrophoresis, precast gels are discarded, thus eliminating the need to clean glass plates.

Figure 3

DNA Sequencing and Genotyping Applications

The Model 4200 (IR2) Automated DNA Analysis System was used in conjunction with the CastAway precast gel to obtain sequence from M13mp19 single-stranded DNA using the M13 (-29) forward primer (Figure 3). The autosequencer called 600 bases with 99.7% accuracy, and data were collected in 2.5 hours (from time of load) using the aforementioned electrophoresis conditions. Read lengths greater than 500 bases are typically achieved for sequence ladders generated on precast gels and analyzed in either the systems 700- or 800-nm channel; this compares to read lengths obtained with manually poured gels (data not shown). The total base number per precast gel is greater than 16,000 when the gel is maximally loaded (i.e., 32
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