Class 6 DNA Arrays BIOMEMS, Fall 2011

Content u Polymerase Chain Reaction or PCR u DNA Detection Process u DNA Micro Arrays u Electronic DNA Arrays u DNA Microarray vs. DNA-Chip u Nanomanipulator

Polymerase Chain Reaction or PCR u Chemical structurs of single stranded DNA: 4 types of Nucleotides in DNA –Adenosine (A) –Guanine (G) –Cytosine (C) –Thymine (T) u Single stranded DNA will form double stranded DNA only with it’s complement: G- C and T-A u Hydrogen Bonding holds strands together

Polymerase Chain Reaction or PCR

u RNA is single stranded; uracyl replaces thymine Polymerase Chain Reaction or PCR

u PCR is an exponential processes (y=e x ) u step 1 - Denaturation (optimal temperature is 94°C): By heating the DNA, the double strand melts and opens to 2 single stranded DNA molecules. u step 2 - Annealing (optimal temperature is 60°C) The single-stranded primers bind to their complementary single-stranded bases on the denaturated DNA. u step 3 - Extension 72°C is the ideal temperature for the Taq polymerase to attach and start copying the template. The result is two new helixes in place of the first.

Polymerase Chain Reaction or PCR u By applying several times this cycle, the quantity of DNA obtained is quickly enough to perform any analysis. Starting with one DNA molecule after just 20 cycles there will be a million copies and after 30 cycles there will be a billion copies. u The taq-polymerase (Thermus aquaticus ) needs ca. 1 min to synthesise 1 kbp. So the synthesis time depends on the length of your product. u The bacterium Thermus aquaticus was first discovered in several springs in the Great Fountain area of the Lower Geyser Basin at Yellowstone National Park.

u ST Lab-On-Chip Miniaturization of PCR Polymerase Chain Reaction or PCR

DNA Detection Process DNA/RNA extraction from the cells or microorganisms Sample preparation  Chemical extraction (alkali)  Mechanical disruption (ultrasonics) DNA/RNA purification  Filters (size exclusion)  Specific adsorption (silica)  Commercial kits Target DNA hybridization to complementary probes on the DNA microarray DNA amplification  PCR (polymerase chain reaction)  Isothermal amplifications (strand displacement)  On-chip amplification Detection  Labeled target or additional reporter probe  Fluorescent detection

NanoChip ® Cartridge Fluidic and electronic interface Electronic DNA Array

Smallpox Yersinia pestis Anthrax Staphylococcus aureus

Concentration & Hybridization Fluorescent Detection Electronic DNA Array

_ _ _ _ _ _ _ _ _ _ _ _ Single base pair mismatch Electronic DNA Array

u Nanogen’s active DNA array (100, 400, 10,000 sites) –Transport –Addressing –Concentration –Stringency u Improvements needed: make much smaller, merging with sample preparation, and avoid desalting while maintaining speed of hybridization reaction 10,000-Site CMOS Chip Electronic DNA Array

 One sample - multiple genes  Multiple samples - one gene  Single site multiplexing Total Flexibility: Electronic DNA Array

Standard NanoChip  CMOS chips All control and sensing is provided by the host system Control, sensing and data storage is on-chip Electronic DNA Array

DNA Chips: SYNTHESIZED Probes are deoxyoligo- nucleotides synthesized on glass by solid-phase DNA synthesis coupled with selectively masked light protection and deprotection [photolithography]. Commercial GeneChip have about 300,000 probes on 1.28 x 1.28 cm surface. Experimental versions exceed 1,000,000 probes per array. Microarray: SPOTTED Probes [0.6 kb kb] are PCR amplified full-length cDNA* sequences. Spotted by ‘robo-arms’ on non-porous, solid support. About 10,000 ‘spots’ on a microscope glass slide. DNA Microarray vs. DNA-Chip * In genetics, complementary DNA (cDNA) is DNA synthesized from an mRNA template in a reaction catalyzed by the enzyme reverse transcriptase

Nanomanipulator Particle less polarizable than medium u Use DC and AC electrokinetics to write with particles:

Before Wash Listeria off After wash blood off Wash blood off Separation: 10 kHz, 10Vpp Separation of Listeria from Whole Blood Nanomanipulator