DNA Stability and Handling Flashcards
How is duplex stability measured?
What does negative ΔG mean?
When is positive ΔG good?
ΔG = ΔH - TΔS
where:
ΔH = change in enthalpy (heat)
T = temperature
ΔS = change in entropy (disorder)
- negative ΔG = stable
- the more negative, the more stable
- negative ΔG = hybridisation favourable
- when want to split up DNA and analyse it
What are two axis on plot and what is dotted line halfway through?
- x-axis: temperature
- y-axis: percentage of dissociated strands (A)
- Tm (melting temperature when we go from associated to dissociated is optimises within reason to be at temperature our bodies are at
- this is an equilibrium
- at lower temp, most is in double helix
- at higher temp, separate strands of DNA has H-bonds been broken
- double to single - melting
- single to double - annealing
What are some factors that affect the stability of the double helix?
- temperature
- salt concentration
- pH
- neighbouring bases
- ion charge
- sequence length
- denaturants
How does neighbouring bases affect double helix?
- due to pi-stacking
- each internal base pair adds stability as they are enthalpically favoured and entropically disfavoured
- GC has more hydrogen bonds so it has a larger ΔG, therefore more stability and increase in melting temperature
Why is DNA double helix more stable than RNA single strand?
- double helix
- more ordered = entropic penalty
- hydrogen bonds and pi stacking = enthalpic gain
- single strand
- less ordered = entropic gain
- hydrogen bonds broken = enthalpic penalty
How do you measure duplex stability?
- add all the ΔG together for each bond
- all calculations are performed in 1M NaCl (salty so stabilises double bond)
How does temp affect duplex stability?
- as increase temp, disorder increases (ΔS), so ΔG gets less (less stable)
- as temp increases, enthalpic contribution decreases and entropic contribution increases
- if want to store DNA for long time, need to keep as cold as possible
How does salt concentration affect duplex stability?
- DNA needs cations to shield the charge of the phosphate groups
- increasing salt conc pushes up melting temp = increase in stability
- M2+ cations are more strongly stabilising (larger and denser) than M+ cations as less ions required to stabilise DNA to same effect
How does pH affect duplex stability?
- DNA must be kept at controlled pH to control protonation and prevent degradation
- reduce pH less than 5
- protonation occurs
- hydrogen bonds are broken
- amines are protonated
- DNA destabilised
- increase pH more than 10
- deprotonation occurs in hydrogen bonds
- creating HB mismatches which will be repulsive
- degradation
- decrease in melting temp
- DNA destabilised
What can some chemicals do to DNA?
When is this useful?
- DNA can be denatured by chemicals, particularly those which compete for the hydrogen bonding sites
- can be used to our advantage when we are separating and analysing a DNA sample
What does DNA look like?
- DNA is a transparent film or tiny quantity of white powder
How would making the sequence longer effect the melting temperature of DNA duplex?
- ends of duplex are relatively destabilising
- so longer sequence = reduces their significance, increase melting temp and stability
How do we handle DNA?
- cannot weigh like a solid so must do everything in solution
- DNA is very soluble in pure water but we use buffers to ensure that we have control over protonation state and prevent degradation
- depurination: starts just below pH7 and DNA in pure water = pH5
What are main 2 things in buffer?
and why are they used?
- mixture of base and it’s conjugate acid
- base:
- tris ((Tris (hydroxymethyl) aminomethane)
- acid:
- acetic acid - good for stabilising enzymes but can overheat and decompose in gels
- boric acid - not great for enzymes as it inhibits them but less overheating in gels
- HCl - used in PCR
- to maintain a particular pH (stabilise pH so DNA doesn’t degrade)
What other three things are common in buffer
and two uncommon things?
COMMON
- water
- distilled and then filtered to a very high purity
- sometimes autoclaved too
- expensive process but vital
- EDTA (e.g. Cu)
- binds stray metal ions which could damage DNA
- salts (salts, potassium, and/or magnesium)
- needed for hybridisation and may be required for enzymatic activity (e.g. Mg2+ in PCR)
UNCOMMON:
- denaturants (urea or formaldehyde)
- ensures bands on gels represent linear single strands
- surfactants
- mop up unwanted greasy matter (cellular material)
