Focal extraction of surface-bound DNA from a microchip using photo-thermal denaturation.
High-throughput, selective extraction of a particular DNA fragment from a mixture of DNA before PCR amplification is becoming increasingly important in the DNA analysis field. Although the latest microchip technology has enabled real-time DNA expression analysis using hybridization between surface-bound probe DNA and sample DNA, the potential of this technology in purification of a small amount of DNA has not been demonstrated. We report here a method for area-selective release and collection of specific DNA, in which an IR laser beam is focused onto surface-bound sample DNA at the target-spotted area to denature hybridized DNA. First, sample DNA labeled with a fluorescent dye was hybridized to a probe DNA immobilized on a chromium-coated chip. A 1053-nm IR laser beam with an intensity of 10-100 mW was then focused on the target area with a spatial resolution of 10 microns, causing the release of the fluorophore-labeled sample DNA as a result of photo-thermal denaturation. Confirmation of the amount of eluted DNA by PCR amplification after collection indicated that more than 10(-20) mol DNA/micron 2 area was eluted from the microchip, representing more than 70% of the chip-bound sample DNA. These results indicate that this method can be applied to the highly sensitive purification of DNA in microchip technology.
Yasuda
K
K
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Japan. cyasuda@mail.ecc.u-tokyo.ac.jp
Okano
K
K
Ishiwata
S
S
D002857:Chromium; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005456:Fluorescent Dyes; D005898:Glass; D007259:Infrared Rays; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008858:Microscopy, Phase-Contrast / Q000295:instrumentation / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D010777:Photochemistry; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D013696:Temperature
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D002857:Chromium