W2L3+4 - PCR Flashcards
Positive Control
Sample known to contain region of interest that you intend to amplify
After PCR should demonstrate only then band that you intend to amplify
Need to know expected size of product`
Negative Control
2 types:
Blank or Contamination Control
- contains all components of a PCR except DNA
- usually water
- ensures that reagents aren’t contaminated
Negative Template Control
- contains DNA that doesn’t contain specific region you are attempting to amplify
- ensures that the PCR is not amplifying DNA non-specifically
Amplification Control
Uses a second set of primers that are not specific to your region of interest however they are specific to a region that you know is present in the sample
Ensures that you have sample in the reaction and that the reaction works
Factors Affecting PCR
[Magnesium]
Polymerase used
Primer design
Annealing temperature
Factors Affecting PCR - [Magnesium]
Mg2+ is a cofactor required for activity of DNA polymerase
Also binds DNA and dNTPs
If too much is present, the stringency of the reaction decreases
Factors Affecting PCR - Primer Design
For successful PCR, primers need to bind to the template DNA
Binding is mediated by H bonding between complementary base pairs
Degree of H bonding is affected by the composition of the primer
Primer composition variables that affect binding:
1. Primer length - longer primers have more nucleotides = more H bonds
2. Nucleic acid composition - C-G pairs have 3 H bonds while A-T pairs have 2
3. Degree of sequence homology - mismatch between bases = less H bonds
Factors Affecting PCR - Annealing Temperature
Degree of H bonding and therefore primer binding is affected by the reaction temperature
The lower the annealing temperature the more likely the primer is to bind, but it will bind with lower specificity (low stringency) and vice versa
Ideal Primer
18-25 bp long
Has a sequence identical to the target region
Has a G-C content of roughly 50%
Has a melting point of roughly 55-60°C
Primers should not be complementary to each other
- results in a primer dimer
Mutagenic PCR
Mutations can be deliberately introduced into PCR products by designing primers that aren’t complementary to the target
Useful for introducing restriction sites
Isothermal PCR
Traditional PCR relies on heat to denature DNA
Some DNA polymerases exhibit strand displacement activity which allows them to denature DNA while amplifying it
This allows amplification reactions to be performed at a single temperature
Benefit - lower equipment cost, can be used outside of lab
Loop Mediated Isothermal Amplification
DNA initially denatured by heat then chilled
Bst polymerase added and the reaction is incubated at a single temperature
Multiple large, complex DNA structures form that can be detected using gel electrophoresis, DNA dyes, pH indicators
Need to use at least 4 primers in a single reaction
