Genotyping by dynamic heating of monolayered beads on a microheated surface.
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.
Russom
Aman
A
Department of Signals, Sensors and Systems, Microsystem Technology, Royal Institute of Technology, Stockholm, Sweden. aman.russom@s3.kth.se
Haasl
Sjoerd
S
Ohlander
Anna
A
Mayr
Torsten
T
Brookes
Anthony J
AJ
Andersson
Helene
H
Stemme
Göran
G
D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D004867:Equipment Design; D005838:Genotype; D006358:Hot Temperature; D056656:Lab-On-A-Chip Devices; D046208:Microchip Analytical Procedures / Q000379:methods; D008863:Microspheres; D008904:Miniaturization; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000032:analysis / Q000235:genetics; D020641:Polymorphism, Single Nucleotide
D009841:Oligonucleotides