Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

✍ Scribed by Ann Wainright; Uyen T. Nguyen; TorLeif Bjornson; Travis D. Boone

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 275 KB
• Volume: 24
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.200305594

✦ Synopsis

Abstract

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™ 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 ΦX174/__Hae__III 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/μL of DNA in 0.1×polymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.