Nucleic Acid Purification : Its Principle and Miniaturization MEC seminar Apr. 13 Ji Youn Lee

mRNA separation module (immobilized oligo-dT) mRNA separation module (immobilized oligo-dT) Hybridization module Target mRNA capture module Fluorescence detection module Inlet I Outlet Inlet II Waste Heating region Lysis of tissue (or cells) and nucleic acid purification

Topics Brief introduction of nucleic acid purification principle Summary of papers about nucleic acid purification on chip

Purification from What? Proteins Enzymes, DNA- or RNA-binding proteins, transcription factors… etc. Enzymes, DNA- or RNA-binding proteins, transcription factors… etc. Salts Buffer change Buffer change Other contaminants Short DNA fragments, dNTPs… etc. Short DNA fragments, dNTPs… etc. Commercially available products PCR cleanup kit, gel elution kit, plasmid, genomic DNA, total RNA, mRNA purification kits PCR cleanup kit, gel elution kit, plasmid, genomic DNA, total RNA, mRNA purification kits

Selective binding to filter (in the presence of the chaotropic salt) Washing with ethanol Elution with low-ionic strength buffer Principle Disruption of the water structure around negatively charged silica, allowing a cation bridge to form. (guanidinium isothiocyanate, sodium iodide, and etc.) When the salt is removed, rehydration of the silica matrix breaks the attraction between the matrix and DNA.

Binding buffer + sample (Chaotropic salts) Washing buffer (Ethanol) Elution buffer (Low salts) Centrifugation Purified nucleic acid solution General Procedure Filter

How Miniaturize? Material packing (silica beads) Incorporation of silica-based resins into micro-flow device Incorporation of silica-based resins into micro-flow device Electrophoresis 23, 727–733 (2002) Electrophoresis 23, 727–733 (2002) Analytical Biochemistry 283, 175–191 (2000) Analytical Biochemistry 283, 175–191 (2000) Pillar structure Using microfabricated silica pillar structures Using microfabricated silica pillar structures

Nucleic Acid Purification Using Microfabricated Silicon Structures Nathaniel C. Cady, Scott Stelick, Carl A. Batt Biosensors and Bioelectronics 19 (2003) (Cornell Univ.)

Summary Objective A microfluidic device to purify bacteriophage lambda DNA and bacterial chromosomal DNA A microfluidic device to purify bacteriophage lambda DNA and bacterial chromosomal DNA Materials A microfabricated channel in which silica-coated pillars were etched A microfabricated channel in which silica-coated pillars were etched Methods DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. followed by washing with ethanol and elution with low-ionic strength buffer.

Summary (continued) Results Surface area Surface area Initial: 0.7 cm 2 Initial: 0.7 cm 2 With pillars: 2.1~4.2 cm 2 With pillars: 2.1~4.2 cm 2 Surface to volume ratio Surface to volume ratio 4200 cm 2 /ml 4200 cm 2 /ml Efficiency Efficiency ~16% ~16% Capacity Capacity 200 ng/2.45 cm 2 = 82 ng/cm2 200 ng/2.45 cm 2 = 82 ng/cm2

Fig. 1 Schematic representation of channels containing microfabricated silica pillars. The spacing between pillars and the pillar width was kept constant at 10  m, while the depth of the channels and height of the pillars could be adjusted between 20 and 50  m. 50  m 10  m

Fig. 2

Fig. 3 DNA elution profile for total DNA from 10 7 E. coli cells. Initial 200 ㎕ wash with TE buffer (fractions 1~4) 10 7 E. coli cells in 100 ㎕ of L6 buffer were loaded (fractions 5~6) Ethanol wash (fractions 7~10) Eluted with TE (fractions 11~16) The error bars represent the standard deviation between three separate experiments.

Fig. 4 DNA and protein concentration profile for DNA purification from 10 7 E. coli cells. Protein (87% removal) DNA