96-well polycarbonate-based microfluidic titer plate for high-throughput purification of DNA and RNA.
We report a simple and effective method for the high-throughput purification of a variety of nucleic acids (NAs) from whole cell lysates or whole blood using a photactivated polycarbonate solid-phase reversible immobilization (PPC-SPRI) microfluidic chip. High-throughput operation was achieved by placing 96 purification beds, each containing an array of 3800 20 microm diameter posts, on a single 3" x 5" polycarbonate (PC) wafer fabricated by hot embossing. All beds were interconnected through a common microfluidic network that permitted parallel access through the use of a vacuum and syringe pump for delivery of immobilization buffer (IB) and effluent. The PPC-SPRI purification was accomplished by condensation of NAs onto a UV-modified PC surface in the presence of the IB comprised of polyethylene glycol, NaCl, and ethanol with a composition dependent on the length of the NAs to be isolated and the identity of the sample matrix. The performance of the device was validated by quantification of the recovered material following PCR (for DNA) or RT-PCR (for RNA). The extraction bed load capacity of NAs was 206 +/- 93 ng for gDNA and 165 +/- 81 ng for TRNA from Escherichia coli. Plate-to-plate variability was found to be 35 +/- 10%. The purification process was fast (<30 min) and easy to automate, and the low cost of wafer fabrication makes it appropriate for single-use applications.
Witek
Małgorzata A
MA
Center for Bio-Modular Multi-Scale Systems, Louisiana State University and Agricultural and Mechanical College, 8000 GSRI Road, Baton Rouge, Louisiana 70820, USA.
Hupert
Mateusz L
ML
Park
Daniel S-W
DS
Fears
Kirby
K
Murphy
Michael C
MC
Soper
Steven A
SA
D000818:Animals; D004247:DNA / Q000097:blood / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000302:isolation & purification; D004586:Electrophoresis / Q000379:methods; D006801:Humans; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D011076:Polycarboxylate Cement / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000302:isolation & purification; D012343:RNA, Transfer / Q000302:isolation & purification; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods
D004269:DNA, Bacterial; D011076:Polycarboxylate Cement; D012333:RNA, Messenger; D012335:RNA, Ribosomal; C005506:polycarbonate; D012313:RNA; D004247:DNA; D012343:RNA, Transfer