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)
What is the equation that is used to make a buffer?
Henderson-Hasselbalch
pH = pKa + log10 ([B]/[BH+])
pH = of buffer solution
pKa = of buffer compound e.g. Tris
[B] = concentration of free base
[BH+] = concentration of conjugate acid
Make a pH 8 Tris/acetate buffer at a concentration of 45 mM
(i.e. How much acid do we need to add ([BH+]) to generate a pH 8 Tris/acetate buffer at 45mM?)
pH = pKa + log10 ([B]/[BH+])
8 = 8.12 + log10 ([B]/[BH+])
-0.12 = log10 ([B]/[BH+])
[B] + [BH+] = 0.045
[B] = 0.045 - [BH+]
10^-0.12 = 10^log10 ([B]/[BH+])
0.759 = [B]/[BH+]
0.759 = 0.045 - [BH+]/[BH+]
0.759 [BH+] = 0.045 - [BH+]
0.759 [BH+] + [BH+] = 0.045
1.759 [BH+] = 0.045
[BH+] = 0.0255 M
[BH+] = 2.55 mM
How is buffer made in practice?
- make a stock solution at 10x concentration and dilute as needed
- translate into grams of pure tris and mL of HCl
- this is an approximation as real values depend on buffer conc, temp and presence of salts
- so still have to measure pH to check
How do you measure concentration of DNA?
- beer lamber law - relationship of light absorption to sample concentration
- A = ϵ c l
- where
- A = absorbance (aka optical density)
- ϵ = absorptivity in M-1cm-1 (constant)
- c = concentration
- l = path length in cm
How can you work out transmission?
How is this related to absorbance?
- T = P/P0
- where:
- P0 = intensity before
- P = intensity after
- A = log (P0/P)
What is ϵ for DNA?
What wavelength do we measure these at and why?
At this wavelength what is conc is absorbance is 1
- absorptivity for DNA varies a little bit depending on what type of DNA we have
- dsDNA = 0.020 (ug/ml)-1 cm-1
- ssDNA = 0.027 (ug/ml)-1 cm-1
- RNA = 0.025 (ug/ml)-1 cm-1
- at wavelength 260 nm
- this is wavelength at which we get highest absorption because this is wavelength at which nucleobases absorb due to their aromatic rings
- A260 = 1 and pathlength is 1
- c = 1/0.02
- c = 50 ug/ml for dsDNA
What instruments are used to measure the concentration of DNA?
- UV-Vis spectrophotometer
- requires 1-3 mL and can only do one sample at a time
- collects whole spectrum
- UV-Vis plate reader
- requires 50 - 300 uL and can do many samples at once
- whole spectrum or single wavelength
- good for DNA
- NanoDrop
- requires 1 uL and sample at a time
- usually single wavelength so quick but limitation of one sample at a time
- sensitive (lot of things can affect value so be careful with cleaning)
Why is it important to be able to measure something small?
as do not want to dilute something too much
