DNA Stability & Handling Flashcards
Equation for ΔG
ΔG = ΔH - TΔS
Change in entropy - (temperature in kelvin multiplied by change in entropy)
Negative ΔG
The more stable it is as hybridisation is favourable
ΔH
Enthalpy
ΔS
Entropy
Why is stability affected by neighbouring bases?
Stability is affected by neighbouring bases due to pi stacking.
How is pi stacking measured?
- In pairs
- Each internal base pair adds stability
- All internal base pairs are enthalpically favoured and entropically disfavoured
ΔG
Gibbs free energy
What happens to the enthalpy of base pairs on the edge?
Pairs on the edge are much more free so they’re enthalpically disfavoured
Why are the internal base pairs entropically disfavoured?
They are more rigid and unable to move so they’re entropically disfavoured.
CG base pair
- CG pair has 3 hydrogen bonds
- Delta G for anything involving this pair, they all have values around -9.
AT base pairs
- Only 2 hydrogen bonds
- Have smaller delta G values
DNA vs RNA duplex stability
Enthalpy & entropy
DNA:
- More ordered = Entropically disfavoured (loss)
- Hydrogen bonding and pi stacking = Enthalpically favoured (gain)
- More ordered because the base pairs are bonded together.
- Due to pie stacking between layers, that stabilises it, as well as hydrogen bonds, we do gain from an enthalpic position.
RNA:
- Less ordered - entropically favoured (gain)
- Hydrogen bonds broken = Enthalpically disfavoured (loss)
- Far less hydrogen bonds
What increases entropic contribution?
Entropic contribution increases with temperature
- As we increase temp we’re increasing contribution of Delta S and you end up with something thats unstable.
- Increasing temperature will cause our DNA to become unstable
What do you need to do it you want to store DNA?
you need to cool it down for as long as possible
What does DNA need cations for?
- DNA needs cations to shield the charge of the phosphate groups.
- If a negative charge pushes thre DNA structure apart and destabilises it then adding a cation can shield this from happening.
M2+ vs M+ cations
M2+ cations are more strongly stabilising than M+ cations
What does increasing the salt concentration do to DNA?
- Increasing the salt content pushes up the melting temperature of DNA
- DNA exists in our body in a salty environment so we want to mimic these conditions
What does increasing Mg or Na do to DNA?
- Increasing the magnetism or sodium increases the stability
- When sodium makes a shift its far smaller than the equivalent impact from magnesium
- Less magnesium is required to make the same impact but it has less of an effect on the temperature
What affect does sodium groups have in DNA?
Sodium exists around phosphate groups in order to stabilise it.
Why do we need fewer Mg compared to Na?
Magnesium is larger and has a higher density of charge so we need fewer of them compared to sodium to get the same level of stability.
Duplex stability in response to neutral pH?
DNA needs to be kept at a controlled pH to control protonation and prevent degradation
Duplex stability in reponse to pH <5
- We see protonation of amines, we’re breaking some of the hydrogen bonds.
- This starts destabilosng the structure.
- If pH is lower enough you could completely change the chemistry
Duplex stability in reponse to pH >10
- The amine groups get deprotonated and the structure starts to repel due to negative charges.
- Some of the hydrogen bonds get broken so it is less stable.
Duplex chemical denaturation
- DNA can be denatured by chemicals, particularly those which compete for the hydrogen bonding sites.
- This can sometimes be used to our advantage when we are separating and analysing a DNA sample.
- This doesn’t happen inside our body but will happen when we take it out
- Urea and Formamide have lots of potential carboxylates and amines that can interact and disrupt hydrogen bonding in DNA as they are small enough, this could potentially dentature our DNA.
Why do we use buffers?
DNA is very soluble in pure water, but we typically use buffers to ensure that we have control over protonation state and prevent degradation.
When does depurination occur?
It starts are just below pH 7
What is a buffer solution?
- It is a mixture of a base and its conjugate acid.
- It is used to maintain a particular pH and regulate.
Acetic acid
- Good for use of enzyme stability
- It can overheat in gels leading to destabilisation
Boric acid
- Inhibits enymes
- Less overheating in gels
Hydrochloric acid
Used in PCR
Water in buffer solution
- Water is distilled and then filtered to a very high purity and sometimes autoclaved too.
- It is sometimes irradiated to kill anything else present.
Common ingredients in a buffer solution
- EDTA: Binds stray metal ions which could damage DNA (e.g. Cu)
- Salts: Sodium, potassium, and/or magnesium as needed for hybridisation and may be required for enzymatic activity (e.g. Mg2+ in PCR)
What is sometimes added to a buffer solution?
- Denaturants if we want to degrade the DNA: Urea or formamide ensures bands on gels represent linear single strands.
- Surfactants are in there as the cellular material is a greasy material (likely found in DNA) which the surfactants remove.
pH of the buffer solution equation
= pKa of the buffer compound + log10 (Concentration of the free base / concentration of the conjugate acid)
What do real buffer solutions depend on?
- Buffer concentration
- Temperature
- Presence of salts
Equation for absorption (optical density)
Beer lambert law
Absorptivity x path length x Concentration of absorbing solution
A = ε c l
I
- Path length
- cm
ε
- Absorptivity
- Constant
- M-1 cm-1
Concentration of absorbing solution (c)
A/(εl)
Transmission (T) equation
T = P / P0
Absorption using P
A = log (P0/P)
UV-Vis spectrometer
Measure concentration of DNA
- Requires 1-3mL
- One sample at a time
- Collects whole spectrum
- Good for more complex solutions
- Good starting points for unknowns
UV-Vis Plate reader
Measuring concentration of DNA
- Requires 50-300uL
- Many samples as once
- Whole spectrum or single wavelength
- Quicker than spectrometer
Nanodrop
Measuring concentration of DNA
- Requires 1uL (smallest volume of sample)
- Good for trace amounts
- One sample at a time
- Usually single wavelength
- Immediately gives a reading
- Incredibly sensitive but too sensitive in some circumstances
- It will generate values for contaminations when this isnt wanted at times
