Class 7

DNA and Protein ArraysBIOMEMS, Winter 2009

Content

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

Polymerase Chain Reaction or PCR

Chemical structurs of single stranded DNA: 4 types of Nucleotides in DNA

– Adenosine (A)– Guanine (G)– Cytosine (C)– Thymine (T)

Single stranded DNA will form double stranded DNA only with it’s complement: G-C and T-A

Hydrogen Bonding holds strands together

Polymerase Chain Reaction or PCR

RNA is usually single stranded in a cell ; uracyl replaces thymine

Polymerase Chain Reaction or PCR

Polymerase Chain Reaction or PCR

PCR is an exponential processes (y=ex )

step 1 - Denaturation (optimal temperature is 94°C): By heating the DNA, the double strand melts and open to single stranded DNA.

step 2 - Annealing (optimal temperature is 60°C) The single-stranded primers bind to their complementary single-stranded bases on the denaturated DNA.

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

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.

The taq-polymerase (Thermus aquaticus ) needs ca. 1 min to synthesise 1 kbp. So the synthesis time depends on the length of your product.

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

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.

TTAACGGGGCCCTTTAAA.....…TTTAAACCCGGGTTT pairs with: AATTGCCCCGGGAAATTT........AAATTTGGGCCCAAA

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.

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

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 probeson 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® CartridgeNanoChip® Cartridge

Fluidic and electronic interface

Electronic DNA Array

Electronic DNA Array

SmallpoxYersinia pestis

Anthrax

Staphylococcusaureus

Concentration & HybridizationConcentration & Hybridization

Fluorescent DetectionFluorescent Detection

Electronic DNA Array

____________

Single base pair mismatch

Electronic DNA Array

Nanogen’s active DNA array (100, 400,

10,000 sites)

– Transport

– Addressing

– Concentration

– Stringency Improvements needed: make much smaller,

merging with sample preparation, and avoid desalting while maintaining speed of hybridization reaction

10,000-Site CMOS Chip10,000-Site CMOS Chip10,000-Site CMOS Chip10,000-Site CMOS Chip

Electronic DNA Array

QuickTime™ and aCinepak decompressor

are needed to see this picture.

QuickTime™ and aCinepak decompressor

are needed to see this picture.

One sample - multiple genes

Multiple samples - one gene

Single site multiplexing

Total Flexibility:

Electronic DNA ArrayElectronic 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 ArrayElectronic DNA Array

DNA Chips: SYNTHESIZED

Probes are 20-25 deoxyoligo-nucleotides synthesized on glassby solid-phase DNA synthesis coupled with selectively maskedlight protection and deprotection[photolithography]. CommercialGeneChip have about 300,000probes on 1.28 x 1.28 cm surface.Experimental versions exceed1,000,000 probes per array.

Microarray: SPOTTED

Probes [0.6 kb - 2.4 kb] are PCR amplified full-lengthcDNA sequences.Spotted by ‘robo-arms’ on non-porous, solid support.About 10,000 ‘spots’ on amicroscope glass slide.

DNA Microarray vs. DNA-Chip

Nanomanipulator

Particle less polarizable than medium

Use DC and AC electrokinetics to write with particles:

Before

Wash Listeria offAfter wash blood off

Wash blood offSeparation: 10 kHz, 10Vpp

Separation of Listeria from Whole BloodSeparation of Listeria from Whole Blood

Nanomanipulator