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

Polymerase Chain Reaction or PCR
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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

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

Polymerase Chain Reaction or PCR
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PCR is an exponential processes (y=e x ) step 1 - Denaturation (optimal temperature is 94 ° C): By heating the DNA, the double strand melts and opens to 2 single stranded DNA molecules.
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
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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.
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
Miniaturization of PCR
 ST Lab-On-Chip

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

Electronic DNA Array
NanoChip
®
Cartridge

Electronic DNA Array

Electronic DNA Array

Electronic DNA Array
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Single base pair mismatch

Electronic DNA Array
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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 Chip

Electronic DNA Array
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One sample - multiple genes
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Multiple samples - one gene
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Single site multiplexing

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

DNA Microarray vs. DNA-Chip
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 Chips: SYNTHESIZED
Probes are 20-25 deoxyoligonucleotides 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.
* In genetics, complementary DNA (cDNA) is DNA synthesized from an mRNA template in a reaction catalyzed by the enzyme reverse transcriptase

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

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