Class 7 DNA and Protein Arrays BIOMEMS, Winter 2009

Content u Polymerase Chain Reaction or PCR u Cells are Molecular Factories u DNA Detection Process u DNA Micro Arrays u Electronic DNA Arrays u DNA Microarray vs. DNA-Chip u Lab on a chip u DNA Chip Preparation u Applications of DNA Arrays u Nanomanipulator u Protein Arrays

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 usually single stranded in a cell ; 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 open to single stranded DNA. 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 large 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.

Polymerase Chain Reaction or PCR u In PCR, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 nucleotides, and they match exactly the beginning and the end of the DNA fragment to be amplified. They direct replication towards each other - the extension of one primer by polymerase then becomes the template for the other, leading to an exponential increase in the target segment. u TTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTT pairs with: AATTGCCCCGGGAAATTT AAATTTGGGCCCAAA u Now, let's say that the above sequences "flank" (are on either end of..) the gene, which includes a long stretch of letters designated as: These are known, absolutely identified to be, the sequence of letters which ONLY flank a particular region of a particular organism's DNA, and NO OTHER ORGANISM'S DNA. This region would be a target sequence for PCR. u The first step for PCR synthesize "primers" of about 20 letters-long, using each of the 4 letters, and a machine which can link the letters together in the order desired - this step is easily done, by adding one letter-at-a-time to the machine (DNA synthesizer). In this example, the primers we wish to make will be exactly the same as the flanking sequences shown above. We make ONE primer exactly like the lower left-hand sequence, and ONE primer exactly like the upper right-hand sequence, to generate: TTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTT AATTGCCCCGGGAAATTT > and: < …TTTAAACCCGGGTTT AATTGCCCCGGGAAATTT AAATTTGGGCCCAAA

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

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