Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices.
We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.
Wainright
Ann
A
ACLARA BioSciences, Mountain View, CA 94043, USA. wainright@pipeline.com
Nguyen
Uyen T
UT
Bjornson
TorLeif
T
Boone
Travis D
TD
D001331:Automation; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods; D007202:Indicators and Reagents; D007364:Intercalating Agents; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D015336:Molecular Probe Techniques / Q000295:instrumentation; D010969:Plastics; D011089:Polydeoxyribonucleotides / Q000737:chemistry* / Q000302:isolation & purification; D015183:Restriction Mapping; D012680:Sensitivity and Specificity
D007202:Indicators and Reagents; D007364:Intercalating Agents; D010969:Plastics; D011089:Polydeoxyribonucleotides; D004247:DNA