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PCR: Polymerase Chain Reaction
Basics & MiniaturizationBIOMEMS, 2010
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PCR: Polymerase Chain Reaction
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Nucleic Acids: DNA/RNA Polymers of Nucleotides
Deoxyribonucleic acid(DNA) Ribonucleic acid(RNA) 4 types of Nucleotides in DNA
Adenosine (A) Guanine (G) Cytosine (C) Thymine (T)
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DNA StructureSingle stranded DNA
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DNA StructureSingle stranded DNA will form double stranded DNA only with it’s complement
G-C
T-A
Hydrogen Bonding holds strands together
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DNA StructureDouble stranded DNA forms a double helix structure.
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DNA in Cells Humans have 46 chromosomes
Choromosomes (30,000 genes)
Gene- codes for a protein
SS DNA
Loci –small section of a gene
Nucleotide
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DNA/RNA analysis
Human Genomic DNA Mutations(SNP), Deletions, Identification(STR)
Organism Detection Pathogens (virus, bacteria)
RNA Gene expression
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DNA Assay: SNP What is a SNP Assay?
Three Steps: DNA purification
Amplification
Detection
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Example: Solid Phase Microspotting
Specific hybridization based assay
Basis of DNA microarrays Method:
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Example: Real-time PCR
Specific hybridization based assay
Fastest Assay Method Method/Limitations
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PCR is an exponential processes
y=ex
e=2.71828281828
What is special about this number?
How PCR Works
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step 1 - Denaturation (optimal temperature is 94°C)By heating the DNA, the double strand melts and open to single stranded DNA.
How PCR Works
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step 2 - Annealing (optimal temperature is 60°C) The single-stranded primers bind to their complementary single-stranded bases on the denaturated DNA.
How PCR Works
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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.
How PCR Works
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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.
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Taq polymerase
The taq-polymerase needs ca. 1 min to synthesise 1 kbp. So the synthesis time depends on the length of your product
Thermus aquaticus
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Thermus aquaticus
The bacterium Thermus aquaticus was first discovered in several springs in the Great Fountain area of the Lower Geyser Basin at Yellowstone National Park
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How Real-Time PCR Works
diagram taken from TaqMan Gold RT-PCR kit protocol (PE Applied Biosystems)
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Real-Time PCR Data
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Standard PCR Thermocyclers
Thermal Uniformity: ±0.5°C within 30 seconds of arrival at 60°C
Ramping Speed: 60: up to 1°C/second, 96: up to 1.4°C/second, 96AgV: up to 2.5°C/second, 16MS: up to 0.5°C/second
PTC-100®Peltier Thermal CyclerMJ Research
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Fast PCR
Heat block cyclers can take hours to do 30- three temperature cycles. This slow rate leads to long lines for the machine and sign up sheets that force you to start your reactions at all hours. The RapidCycler™ can complete a 30 cycle reaction in less than 10 minutes. Finally, a machine that can keep up with the speed of the biochemistry. No more waiting in line. You can throw away those sign up sheets.
Idaho Technologies
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Fast PCR
The “Core” of Cepheid’s Amplification SystemsReal Time Thermal Cycling
16 independently programmable reaction sites
Faster and more sensitive than reference systems
4-color real time optical detection in each site
1-6 processing blocks
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Miniaturization of PCR ST Lab-On-Chip