Microfluidics for DNA Analysis

1 1EE C245 Microfluidics for DNA AnalysisDr. Thara SrinivasanLecture 19 Picture credit: Nanogen2U. Srinivasan EE C245 Lecture Outline Reading from reader Mastrangelo, C. H. Microfabricated Devices for Genetic Diagnostics, (1998) pp. 1769-87. Khandurina, J. et al., Bioanalysis in microfluidic Devices, (2002) pp. 159-83. Zhang, L., et al., Microchip Electrophoresis-Based Separation of DNA, (2003) pp. 1645-54. Today s Lecture DNA and Analysis Methods Scaling in Microfluidics Survey of Microfabricated Chips23U. Srinivasan EE C245 DNASBP5 3 Genetic information is stored in chromosomes as long strings of DNA grouped as genes In humans, 46 chromosomes are 50 - 400 106 DNA units long (compared to 4 106 for E.)

2 Coli) Units of DNA are nucleotides, consisting of: A base, a sugar and a phosphate bridge Sugar linkage has directionality, 5 and 3 ends Four bases: adenine, thymine, guanine, and cytosine Bases hydrophobic, backbone hydrophilic Single-stranded DNA attaches to complementary strand (G-C, A-T)4U. Srinivasan EE C245 DNA Analysis DNA is extracted from cell nucleus and purified Break cell membranes using detergent Remove cell debris, proteins, enzymes DNA assays Detect specific fragments in fingerprint pattern-matching mode Sequence DNA fragment for base pair order of fragment Analysis tools Chemical amplification Restriction digestion Electrophoretic separation Sanger sequencing process Hybridization Fluorescence visualization35U.

3 Srinivasan EE C245 AmplificationAnimations at: Polymerase chain reaction Double-stranded DNA denatured, 95 C Primers attach (anneal) to strands, flanking section to be amplified, 50-65 C Taq enzymes attach to primer sites and synthesize new strands from bases in solution, 72 C Repeat cycle 20-30 times to get effective amplification Macroscopic thermalcyclers need 90 min per amplification6U. Srinivasan EE C245 Excitation maximumEmission maximumDetection Fluorescent labelingwith molecules which emit light when excited allows extremely sensitive visualization of fragment Intercalating dye: ethidium bromide Single fluorophore: fluorescein Excitation With UV laser-induced fluorescence, emission signal must be separated from excitation; requires confocal microscope Electrochemiluminescence (ECL) uses Ru(bpy)2+3 end label, can be detected with conventional CCD47U.

4 Srinivasan EE C245 Cutting Restriction digestionis fragmentation of DNA Use restriction nuclease enzymes to cleave DNA at specific locations (can recognize specific sequences of 4-8 bases) Size distribution of restriction fragments can fingerprint DNA moleculeMolecular Biology of the Cell8U. Srinivasan EE Pasting Hybridizationis hydrogen bonding of two complementary single strands of DNA Occurs at specific T and salinity conditions In analyses, known strand is probe, other is unknown and binding indicates match Recognition not perfect, single base mismatches occur DNA probes immobilized on surface using linker make pixels for microarrays Microarray pattern matchingACGTACCGTAGCGTATCGTAAGCAT59U.

5 Srinivasan EE C245 Separation Electrophoresis to separate DNA fragments based on size Mobility EPdepends on fragment size and charge and mobile phase DNA fragments in solution are (-) charged and have constant charge to length ratio Additional molecular sieving matrixes are needed to separate DNAbased on length. Fragments drift in race track where separation is L = EP Et Separation resolution important10U. Srinivasan EE C245 Macroscale Separations Macroscale gels Thin multilane slabs; preparation is labor-intensive V up to 2 kV over 20-100 cm Joule heating limits E to 5-40 V/cm Good separation may require hours Capillary electrophoresis Capillaries 10-300 m in diameter, 50 cm long Increased surface to volume ratio and faster heat dissipation permits higher field use (up to kV/cm) Good separation in < 1 hour Use of confocal laser-induced fluorescenceAgilentTechnologies611U.

6 Srinivasan EE C245 Sequencing Sanger method Combine PCR and electrophoretic separations Duplication of DNA fragment starts at primer location, as in But in addition to nucleotides in solution, also add small amount ofdideoxy nucleotides (ddNTP s) of one type (ddA, ddC, ddG, or ddT). When ddNTP is captured, growing strand terminates, resulting ..complementary strand fragments terminated at all possible positions for each base 12U. Srinivasan EE C245 Sequencing Four-color sequencing Carry out four separate reactions, one for each base. Electrophoretically separate each sample Superimpose results to read out fragment sequence713U.

7 Srinivasan EE C245 Today s Lecture DNA and Analysis Methods Scaling in Microfluidics Survey of Microfabricated Chips14U. Srinivasan EE C245 Miniaturization Benefits Benefits Reagent consumption ~ [s3] Miniscule reaction volumes reduce reagent cost. Heat transfer~ [s2] Surface phenomena Mass transfer ~ [s2] Reduced Analysis times, with minimum assay time limited by speed of enzyme (30-100 bp/s) Flow is laminar Electroosmotic flow for valveless systems ~ [s2] Capillary flow ~ [s1] Separation efficiency ~ [s-2] Injection volume well-defined815U. Srinivasan EE C245 Miniaturization Issues Issues Detection limit ~ [s3], S/N degraded as [s3] unless detector area scales with sample [s1] Pressure flows ~ [h3] Other surface phenomena ~ [s2], [s1] Wall adsorption effects and sample evaporation [s2], capillary forces [s1]16U.

8 Srinivasan EE C245 Microfluidics Fabrication Fabrication Batch fabrication Microchip cost ~ [s2], but limited by package cost Parallelization to arrays easy Portability increased Less need for external pumps, detection equipment917U. Srinivasan EE C245 Scaling and MicrofluidicsMastrangelo18U. Srinivasan EE C245 Scaling and Mixing by Diffusion Mixing by diffusion For channels 1 mm wide and flow velocities 1 cm/s, Re is low and flow is laminar Time required to travel distance xby diffusion is x2/2D For channel width of 70 m and velocity 1 cm/s, fluorescein (D= 3 10-6cm2/s) will take 2 s to mix over channel length of 2 mm Upper limit of 100 m width for channels1019U.

9 Srinivasan EE C245 Scaling and Diffusion Effects While being carried by electroosmosis and drifted by electrophoresis, a sample slab can spread out in width due to diffusionDxwSStransitStransitLEDLUDLWD widthtogrowsslabmalinfinitesiULtimein ===0min0, If initial slab is smaller than Wmin, separation is limited only by diffusionLsU020U. Srinivasan EE C245 Scaling and Separation Efficiency Separation efficiency Number of theoretical plates, N, per unit time []][1,,,2,22,22222 = == ====sdtNLdtVLULtdLVDVNEDLDtLNEKiEKxEKxx Resolution parameter, R Peak capacity, n Signal to noise ratio, SNRlLnlRx =~ 1121U. Srinivasan EE C245 Today s Lecture DNA Analysis methods Scaling in Microfluidics Survey of microfabricated devices22U.

10 Srinivasan EE C245 Chip Electrophoresis Capillary electrophoresis (CE) on-chip First demonstrations in 90 s, Manz group (Imperial College, London) and Harrison group (Univ. of Alberta) Separation 100 faster than slab gels, 10 faster than CE CE chips Material ~ glass or plastic Electrodes ~ metal pins inserted into wells or patterned conductive layer Separation medium ~ chips filled withunpolymerized liquids are reusable Layout ~ offset double-T Detection ~ confocal fluorescence microscope focused at single spotCaliper Technologies1223U. Srinivasan EE C245 Parallelization of CE using arrays for high throughput 384-channels radial microplate for genotyping analyses in <7 min with >98% success96-channel wafer, Mathies group UCBA rray CE24U.