DNA Stability & Handling

Question 1

Equation for ΔG

Answer 1

ΔG = ΔH - TΔS
Change in entropy - (temperature in kelvin multiplied by change in entropy)

Question 2

Negative ΔG

Answer 2

The more stable it is as hybridisation is favourable

Question 3

ΔH

Answer 3

Enthalpy

Question 4

ΔS

Answer 4

Entropy

Question 5

Why is stability affected by neighbouring bases?

Answer 5

Stability is affected by neighbouring bases due to pi stacking.

Question 6

How is pi stacking measured?

Answer 6

• In pairs
• Each internal base pair adds stability
• All internal base pairs are enthalpically favoured and entropically disfavoured

Question 7

ΔG

Answer 7

Gibbs free energy

Question 8

What happens to the enthalpy of base pairs on the edge?

Answer 8

Pairs on the edge are much more free so they’re enthalpically disfavoured

Question 9

Why are the internal base pairs entropically disfavoured?

Answer 9

They are more rigid and unable to move so they’re entropically disfavoured.

Question 10

CG base pair

Answer 10

• CG pair has 3 hydrogen bonds
• Delta G for anything involving this pair, they all have values around -9.

Question 11

AT base pairs

Answer 11

• Only 2 hydrogen bonds
• Have smaller delta G values

Question 12

DNA vs RNA duplex stability

Enthalpy & entropy

Answer 12

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

Question 13

What increases entropic contribution?

Answer 13

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

Question 14

What do you need to do it you want to store DNA?

Answer 14

you need to cool it down for as long as possible

Question 15

What does DNA need cations for?

Answer 15

• 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.

Question 16

M2+ vs M+ cations

Answer 16

M2+ cations are more strongly stabilising than M+ cations

Question 17

What does increasing the salt concentration do to DNA?

Answer 17

• 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

Question 18

What does increasing Mg or Na do to DNA?

Answer 18

• 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

Question 19

What affect does sodium groups have in DNA?

Answer 19

Sodium exists around phosphate groups in order to stabilise it.

Question 20

Why do we need fewer Mg compared to Na?

Answer 20

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.

Question 21

Duplex stability in response to neutral pH?

Answer 21

DNA needs to be kept at a controlled pH to control protonation and prevent degradation

Question 22

Duplex stability in reponse to pH <5

Answer 22

• 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

Question 23

Duplex stability in reponse to pH >10

Answer 23

• 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.

Question 24

Duplex chemical denaturation

Answer 24

• 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.

Question 25

Why do we use buffers?

Answer 25

DNA is very soluble in pure water, but we typically use buffers to ensure that we have control over protonation state and prevent degradation.

Question 26

When does depurination occur?

Answer 26

It starts are just below pH 7

Question 27

What is a buffer solution?

Answer 27

- It is a mixture of a base and its conjugate acid. - It is used to maintain a particular pH and regulate.

Question 28

Acetic acid

Answer 28

* Good for use of enzyme stability * It can overheat in gels leading to destabilisation

Question 29

Boric acid

Answer 29

- Inhibits enymes - Less overheating in gels

Question 30

Hydrochloric acid

Answer 30

Used in PCR

Question 31

Water in buffer solution

Answer 31

- Water is distilled and then filtered to a very high purity and sometimes autoclaved too. - It is sometimes irradiated to kill anything else present.

Question 32

Common ingredients in a buffer solution

Answer 32

- 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)

Question 33

What is sometimes added to a buffer solution?

Answer 33

- 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.

Question 34

pH of the buffer solution equation

Answer 34

= pKa of the buffer compound + log10 (Concentration of the free base / concentration of the conjugate acid)

Question 35

What do real buffer solutions depend on?

Answer 35

* Buffer concentration * Temperature * Presence of salts

Question 36

Equation for absorption (optical density) | Beer lambert law

Answer 36

Absorptivity x path length x Concentration of absorbing solution | A = ε c l

Question 37

I

Answer 37

- Path length - cm

Question 38

ε

Answer 38

- Absorptivity - Constant - M-1 cm-1

Question 39

Concentration of absorbing solution (c)

Answer 39

A/(εl)

Question 40

Transmission (T) equation

Answer 40

T = P / P₀

Question 41

Absorption using P

Answer 41

A = log (P₀/P)

Question 42

UV-Vis spectrometer | Measure concentration of DNA

Answer 42

* Requires 1-3mL * One sample at a time * Collects whole spectrum * Good for more complex solutions * Good starting points for unknowns

Question 43

UV-Vis Plate reader | Measuring concentration of DNA

Answer 43

* Requires 50-300uL * Many samples as once * Whole spectrum or single wavelength * Quicker than spectrometer

Question 44

Nanodrop | Measuring concentration of DNA

Answer 44

* 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