1EE C245Microfluidics for DNA AnalysisDr. Thara SrinivasanLecture 19Picture credit: Nanogen2U. Srinivasan ©EE C245Lecture 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 C245DNASBP5’3’• Genetic information is stored in chromosomes as long strings of DNA grouped as genes• In humans, 46 chromosomes are 50 - 400 ×106DNA units long (compared to 4 ×106 for E. 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 C245DNA 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. Srinivasan ©EE C245AmplificationAnimations at: http://www.dnalc.org/resources/BiologyAnimationLibrary.htmhttp://bldg6.arsusda.gov/pberkum/Public/sarl/cregan/pcr.gif• 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 C245Excitation maximumEmission maximumDetection• Fluorescent labeling with 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. Srinivasan ©EE C245Cutting• Restriction digestion is 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 C245…and Pasting• Hybridization is 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. Srinivasan ©EE C245Separation• Electrophoresis to separate DNA fragments based on size• Mobility µEPdepends 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 = ∆µEP Et• Separation resolution important10U. Srinivasan ©EE C245Macroscale 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 µm in diameter, 50 cm long• Increased surface to volume ratio and faster heat dissipation permits higher field use (up to 1.2 kV/cm)• Good separation in < 1 hour• Use of confocal laser-induced fluorescenceAgilentTechnologies611U. Srinivasan ©EE C245Sequencing• Sanger method• Combine PCR and electrophoretic separations• Duplication of DNA fragment starts at primer location, as in PCR…• 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 in…• …complementary strand fragments terminated at all possible positions for each base 12U. Srinivasan ©EE C245Sequencing• Four-color sequencing• Carry out four separate reactions, one for each base. • Electrophoretically separate each sample• Superimpose results to read out fragment sequence713U. Srinivasan ©EE C245Today’s Lecture• DNA and Analysis Methods • Scaling in Microfluidics• Survey of Microfabricated Chips14U. Srinivasan ©EE C245Miniaturization 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 C245Miniaturization 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. Srinivasan ©EE C245Microfluidics 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 C245Scaling and MicrofluidicsMastrangelo18U. Srinivasan ©EE C245Scaling 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 x by diffusion is x2/2D• For channel width of 70 µm and velocity 1 cm/s, fluorescein