Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips.
We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of approximately 7.6 +/- 1.6 microg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 +/- 5%. The immobilization and purification assay using this PPC microchip could be performed within approximately 25 min as follows: (i) DNA immobilization approximately 6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption approximately 6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for 'reactivation' of the PC surface with UV light.
Witek
Malgorzata A
MA
Center for Biomodular Multi-Scale Systems, Louisiana State University, Baton Rouge, LA 70803, USA.
Llopis
Shawn D
SD
Wheatley
Abigail
A
McCarley
Robin L
RL
Soper
Steven A
SA
D002021:Buffers; D004247:DNA / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000097:blood / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D023281:Genomics / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D011076:Polycarboxylate Cement / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D014466:Ultraviolet Rays
D002021:Buffers; D004269:DNA, Bacterial; D011076:Polycarboxylate Cement; C005506:polycarbonate; D004247:DNA