PCR
Steps, components, purpose
Sequencing Technologies
Number of Products at each PCR Cycle
Electrophoresis
Good: Very simple method to separate DNA fragments
Bad: Possible errors due to variability in gel and optical system.
Bad: Possible errors due to variability in gel and optical system.
DNA Fragment Postion
Description
Spread in the DNA
Know the factors
Ladders
Sample of known DNAs
Bunch of different sizes
Compare to your samples
Bunch of different sizes
Compare to your samples
Genotypic Errors
Design Challenges of PCR
To detect geneic polymorphisms and mutations, need a way to:
How to Create Many Many Copies of DNA
PCR
Requires DNA to be etracted from the cell (in vitro/glass)
Requires DNA to be etracted from the cell (in vitro/glass)
How to Separate Strands
Heat to separate strands.
How to Isolate the Gene
Oligonucleotide Primers
How to Copy the Gene
DNA Polymerase and Nucleotides
How Differentiate between mutations/Polymorphisms
Separate DNA by size (Electrophoresis)
Base pair resolutioon for application
Use known gene sequences - diagnosis not discvery
Base pair resolutioon for application
Use known gene sequences - diagnosis not discvery
Purpose
Applications of PCR
Polymorphisms and Mutations
- Cystic Fibrosis
-Disease susceptibility
-Disease susceptibility
Infectious Organisms
- HIV, HPV, Hep B & C
Other
Paternity
Food Industry
Food Industry
1. Obtain a DNA Sample
Extract cells contining chromosomes
2. Prepare Sample for PCR
Extract nucleic acids through centrifugion
PCR Ingredients
DNA Polymerase (TAQ)
Primers
Deoxynucleotides
MgCl2 (to bind the primers)
Primers
Deoxynucleotides
MgCl2 (to bind the primers)
Proteins would Denature with Heat
First they solved this by adding proteins during every thermal cycle.
Next they discovered TAQ polymerase, that can withstand high temperatures.
Next they discovered TAQ polymerase, that can withstand high temperatures.
3. Thermal Cycles
Heat: Denature strands of DNA
Cool: Primers anneal to DNA
Warm: DNA Polymerase attaches and synthesizes
Cool: Primers anneal to DNA
Warm: DNA Polymerase attaches and synthesizes
Steps
Description
Formula
Generation: n
DNA Copies: 2^n
Something:
Product:
DNA Copies: 2^n
Something:
Product:
PCR Amplification
Errors are increased because of PCR amplification
Final Calculations
x/d = C/m
d = distance
C = constant determined by experimental set up
d = distance
C = constant determined by experimental set up
Set up
There are numerous factors you can control like force, distance between electric nodes, etc.
Purpose
Each fragment must be separated to identify pmisms and mutations
Steps
1. DNA fragments placed in a gel (agarose)
2. Gel subjected to electric field
3. Negatively Charged DNA fragments move toward positive electrode
4. Smallest fragments travel the farthest from their original location, largest remain the closest.
2. Gel subjected to electric field
3. Negatively Charged DNA fragments move toward positive electrode
4. Smallest fragments travel the farthest from their original location, largest remain the closest.
Distance vs. Size
Distance traveled is inversely proportional to size of fragment
Note
Harder to distinguish between larger lengths which do not travel as far
Errors
Lines are bands, there is a cumulative errors. Bands sometimes overlap too,
Pricision
-Gel mut be highly homogeneous (pure)
-Optics (camera) must be good)
- Long migration period (long gels)
- Fragments should be short
-Optics (camera) must be good)
- Long migration period (long gels)
- Fragments should be short