Genetics - Gene Analysis Flashcards
Why is DNAP not used in PCR?
Derives from mesophilic bacteria having optimal temperature of 37-40C
How does temperature disrupt enzymatic reactions?
Disruption of activation energy barrier for activity
Activation Energy
This is the energy of the transition state for a given reaction relative to initial state.
How does thermal energy effect catalysis?
Increases rate of reaction by overcoming activation barrier however denatures the protein.
Why does denaturing of the protein occur with temperature?
Disruption of bonds maintaing 3D structure required for activity, like hydrogen bonds, van der Waals force and hydrophobic interactions.
How does Taq polymerase bypass denaturation?
Containing of disulfide bonds and salt bridges.
Disulfide bonds
Covalent interactions between sulfur atoms of two cysteine residues
Salt bridges
Electrostatic interactions between two oppositely charged groups, being an anionic carboxylate and cationic ammonium
Isoelectric Point
This is the point at which the molecule has no net electrical charge.
What is the first step in PCR?
94C temperatures increasing systems energy, breaking hydrogen bonds, DNA strands denature with increased entropy.
What is Gibbs Free Energy related to?
Enthalpy and Entropy
Entropy
This is the measure of the number of ways in which energy can be distributed among the systems particles, or their number of possible arrangements
What does positive enthalpy equate to?
Energy is needs to be added to break hydrogen bonds.
What does positive entropy lead to?
Indicates increase in disorder dominates the thermodynamics, with an overall negative G, meaning denaturaiton is spontaneous.
What is the second step in PCR?
Exothermic annealing of primers
Why is the second PCR step exothermic?
H bonds formed are stronger than the ones broken in denaturation.
Why is entropy small in second PCR step?
Primer/Template bind is ordered compared to ssDNA, G being determined by enthalpy(negative, menaing reaction spontanoeus.
What is the third step in PCR?
Primer elongation with Taq polymerase being exothermic due to negative enthalpy change.