High-throughput asymmetric-PCR SSCP analysis using well-controlled temperature conditions.

Single-stranded conformational polymorphism (SSCP) analysis is useful for mutation detection and polymorphism identification. To realize high-throughput SSCP analysis using a high-intensity electric field, the gel temperature must be well-controlled. We have developed an electrophoretic system having a pair of glass plates with an efficient temperature-controlling apparatus on each side of the gel. The gel temperature is controlled by estimating the temperature difference between the gel and the glass plates, based on a heat-transfer model. Distinctive patterns of asymmetric-PCR SSCP gel electrophoresis were obtained with this system. The patterns showed high reproducibility for an electric field ranging from 40 V/cm to 160 V/cm, as long as the same gel temperature was used. Electric-field strength had little effect on the SSCP patterns. Use of the highest electric field reduced the run time to about 100 min.