Nucleic Acid Purification

: Its Principle and Miniaturization

MEC seminar2004. Apr. 13

Ji Youn Lee

mRNA separation module

(immobilized oligo-dT)

mRNA separation module

(immobilized oligo-dT)Hybridization moduleHybridization module

Target mRNA capture module

Target mRNA capture module

Fluorescence detection module

Fluorescence detection module

Inlet IInlet I

OutletOutlet

Inlet IIInlet II

WasteWaste

Heating region

Lysis of tissue (or cells) and nucleic acid purifica

tion

Lysis of tissue (or cells) and nucleic acid purifica

tion

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 fEnzymes, DNA- or RNA-binding proteins, transcription f

actors… etc.actors… etc. Salts

Buffer changeBuffer change Other contaminants

Short DNA fragments, dNTPs… etc.Short DNA fragments, dNTPs… etc.

Commercially available products PCR cleanup kit, gel elution kit, plasmid, genomic DNPCR cleanup kit, gel elution kit, plasmid, genomic DN

A, total RNA, mRNA purification kitsA, 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 Centrifugation

Centrifugation

Purified nucleic acid solution

General Procedure

Filter

How Miniaturize?

Material packing (silica beads) Incorporation of silica-based resins into micro-Incorporation of silica-based resins into micro-

flow deviceflow device Electrophoresis 23, 727–733 (2002)Electrophoresis 23, 727–733 (2002) Analytical Biochemistry 283Analytical Biochemistry 283, , 175–191 (2000)175–191 (2000)

Pillar structureUsing microfabricated silica pillar structuresUsing microfabricated silica pillar structures

Nucleic Acid Purification Using Microfabricated Silicon Structures

Nathaniel C. Cady, Scott Stelick, Carl A. BattBiosensors and Bioelectronics 19 (2003) 59-66

(Cornell Univ.)

Summary Objective

A microfluidic device to purify bacteriophage lambda DA microfluidic device to purify bacteriophage lambda DNA and bacterial chromosomal DNANA and bacterial chromosomal DNA

Materials A microfabricated channel in which silica-coated pillars A microfabricated channel in which silica-coated pillars

were etchedwere etched Methods

DNA was selectively bound to these pillars in the presDNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate,ence of the chaotropic salt guanidinium isothiocyanate,

followed by washing with ethanol and elution with low-ifollowed by washing with ethanol and elution with low-ionic strength buffer.onic strength buffer.

Summary (continued)

ResultsSurface areaSurface area

Initial: 0.7 cmInitial: 0.7 cm22

With pillars: 2.1~4.2 cmWith pillars: 2.1~4.2 cm22

Surface to volume ratioSurface to volume ratio4200 cm4200 cm22/ml/ml

EfficiencyEfficiency~16%~16%

CapacityCapacity200 ng/2.45 cm200 ng/2.45 cm22 = 82 ng/cm2 = 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 m10 m

Fig. 2

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.

Fig. 4

DNA and protein concentration profile for DNA purification from 107 E. coli cells.

Protein(87% removal)

DNA