W2L3+4 - PCR

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`

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

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

[Magnesium] Polymerase used Primer design Annealing temperature

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

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

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

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

Mutations can be deliberately introduced into PCR products by designing primers that aren’t complementary to the target Useful for introducing restriction sites

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

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