Genotyping by dynamic heating of monolayered beads on a microheated surface

Electrophoresis. 2004 Nov;25(21-22):3712-9. doi: 10.1002/elps.200406065.

Abstract

A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for single-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 4 degrees C-6 degrees C/min compared to earlier reported values of 2 degrees C-3 degrees C/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 microm 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.

MeSH terms

  • Alleles
  • DNA Mutational Analysis / methods*
  • Equipment Design
  • Genotype
  • Hot Temperature*
  • Lab-On-A-Chip Devices*
  • Microchip Analytical Procedures / methods
  • Microspheres
  • Miniaturization
  • Nucleic Acid Denaturation
  • Nucleic Acid Hybridization
  • Oligonucleotides / analysis
  • Oligonucleotides / genetics
  • Polymorphism, Single Nucleotide*

Substances

  • Oligonucleotides