class 6 dna arrays biomems, fall 2011. content u polymerase chain reaction or pcr u dna detection...
Post on 20-Dec-2015
214 views
TRANSCRIPT
Content
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
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
PCR is an exponential processes (y=ex )
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
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.
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
Concentration & HybridizationConcentration & Hybridization
Fluorescent DetectionFluorescent Detection
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
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
* 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
Use DC and AC electrokinetics to write with particles: