Electrophoresis, Vol.25, No.21-22, 3712-3719, 2004
Genotyping by dynamic heating of monolayered beads on a microheated surface
A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for sing le-nucleotide polymorphism analysis is presented. Chips with integrated heater and temperature sensors for open-surface DNA analysis were microfabricated. Microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp was employed to create monolayers of immobilized beads on the surface of the chip. This chip allows fast, well-controllable temperature ramping. The temperature distribution was homogeneous over the entire heater area. All three possible variants of an SNP site of a synthesized oligonucleotide were accurately scored using the bead-based DASH approach. Our assay has a nonoptimized temperature ramping rate of 4degreesC-6degreesC/min compared to earlier reported values of 2degreesC-3degreesC/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 mum was achieved. Our bead-based DASH approach has enabled a dramatic volume reduction and is a step towards developing a cost-effective high-throughput DASH method on arrays of single beads.
Keywords:beads;dynamic allele-specific hybridization;microcontact printing;microheater;miniaturization;single-nucleotide polymorphism