High-speed, multicolor fluorescent two-dimensional gene scanning

Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensiv e multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High-throughput application of TDGS is li mited by the prolonged time periods necessary to complete the second-dimens ion electrophoretic separation step-denaturing gradient gel electrophoresis - and the current need for gel staining. To address these problems, we cons tructed a high-voltage, automatic, two-dimensional electrophoresis system a nd used this in combination with thinner gels to reduce two-dimensional ele ctrophoresis time about 80%. Instead of gel staining, we used three differe nt fluorophores to simultaneously analyze three samples in the same gel. Th ese improvements greatly increase TDGS speed and throughput and make the me thod highly suitable for large-scale single-nucleotide polymorphism discove ry and genetic testing.