Nucleic Acid Purification : Its Principle and Miniaturization

1. Nucleic Acid Purification: Its Principle and Miniaturization MEC seminar 2004. Apr. 13 Ji Youn Lee

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

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

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

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

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

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

9. Nucleic Acid Purification Using Microfabricated Silicon Structures Nathaniel C. Cady, Scott Stelick, Carl A. Batt Biosensors and Bioelectronics 19 (2003) 59-66 (Cornell Univ.)

10. Summary • Objective • A microfluidic device to purify bacteriophage lambda DNA and bacterial chromosomal DNA • Materials • 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, • followed by washing with ethanol and elution with low-ionic strength buffer.

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

12. 10 m 10 m 50 m 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.

13. Fig. 2

14. Fig. 3 • DNA elution profile for total DNA from 107 E. coli cells. • Initial 200 ㎕ wash with TE buffer (fractions 1~4) • 107 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.

15. Fig. 4 Protein (87% removal) DNA DNA and protein concentration profile for DNA purification from 107 E. coli cells.