1. Class 6 DNA Arrays BIOMEMS, Fall 2011

2. 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

3. 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

4. Polymerase Chain Reaction or PCR

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

6. 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.

7. 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.

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

9. 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

10. NanoChip ® Cartridge Fluidic and electronic interface Electronic DNA Array

11. Smallpox Yersinia pestis Anthrax Staphylococcus aureus

12. Concentration & Hybridization Fluorescent Detection Electronic DNA Array

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

14. 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

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

16. 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

17. DNA Chips: SYNTHESIZED Probes are 20-25 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 - 2.4 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

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

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