Lecture 2 - DNA Stability Flashcards
what three interactions is the delta G value of a DNA double helix composed of
(also contributing to DNA stability)
the hydrogen bonds between bases
pi stacking of rings in bases
electrostatic repulsion of phosphate groups
is the double helix of DNA
a) enthalpically favoured or disfavoured
b) entropically favoured or disfavoured
a) favoured due to hydrogen bonds and pi stacking
d) disfavoured (the helical structure and pi stacking makes it more ordered)
is the single strands in RNA
a) enthalpically favoured or disfavoured
b) entropically favoured or disfavoured
a) disfavoured bonds are broken
b) favoured = less ordered than double helix in DNA
how do you calculate delta G for a duplex
what solution are these values measured in and why
adding the detla G value of the nearest neighbouring pair in the sequence (5’-3/3’-5’)
measured in a 1M NaCl solution = salty stabilising the double helix and base pairs
how does a change in temperature affect the duplex stability
hence how is DNA stored
increase in temp decreases stability - we see an increase in delta G (become less negative)
strands become separated as bonds break - system become less ordered and we see an increase in entropy hence a decrease in delta G
hence DNA is stored in the fridge or freezer to prevent this denaturing and destabilisation caused by an increase in temp
what is the Gibbs free energy equation
delta G = delta H - Txdelta S
what is the melting temperature
the temp where half of the DNA strands in the duplex are denatured to single strands
how does a change in salt concentration affect the melting temperature
why does this happen
where can these salts be found
an increase in concentration increase the melting temperature but this gets to a point where it plateaus
increased melting temp corresponds to an increased stability
salt solutions allow metal ions to be present around the DNA
the +ve charge on the metal ions e.g Na+ or Mg2+ is able to stabilise the -ve charge from the phosphate groups preventing electrostatic repulsion
naturally in the body = stable environment for DNA
is a higher or lower concentration of magnesium ions compared to sodium needed to stabilise DNA
why
lower
because Mg ions are 2+ whereas sodium are 1+, these 2+ are more strongly stabilising than the 1+
the 2+ can also re-salt in bridging = further stabilisation
what pH does DNA need to be controlled at and why does this pH need to be controlled
a neutral pH is where DNA is most stable
to prevent DNA degradation by controlling the protonation of components within the DNA structure
what happens when the pH surrounding DNA is >10
at a high pH (alkali solution) deprotonation can occur resulting in negative charges on the Oxygen or Nitrogens involved in hydrogen bonding between base pairs
results in hydrogen bonds being broken = decrease in stability
negative charges reside where deprotonation has occurred
what happens when the pH surrounding DNA is < 5
at a low pH (acidic solution) the oxygen or nitrogen’s can become protonated as this is a favourable process
results in hydrogen bonds being broken = decrease in stability
a positive charge where the protonation occurs
a low enough pH can entirely change the structure and case the rings to be broken - especially in bases with 2 ring structures
how can DNA be affected by other chemicals
give two chemicals that may result in this and give two reasons why
when can this be used to our advantage
DNA can become denatured by chemicals that compete for the hydrogen bonding sites
urea and formamide - these have a lot of available HBA and HBD to disrupt the bonding between bases in DNA, they are also small enough to get into the DNA structure
when wanting to separate and analyse a DNA sample
what is the name of the tubes used in DNA analysis
eppendorf tubes
how small is the amount of DNA we deal with in analysis
samples on the picogram or microgram scale = tiny tiny amounts
the human genome = 3.59 pg
how is DNA handled when it is being analysed
in solution as it can’t be weighed like a chemical reagent
use micropipettes
how is DNA normally put into a solution
using pure water as DNA is very soluble in this
what tends to be added to a DNA solution and why
a buffer solution to control and maintain the pH = creating stabilising conditions for enzyme activity and DNA stability
preventing DNA degradation due to the change in protonation state than can result from a change in pH
keep at pH 7 (pure water tends to be around pH 5 so using buffers is good)
what is a buffer solution made out of (3 components)
a mixture of a base and it’s conjugate acid, an acid and water
(the conjugate acid of a base is where a hydrogen is accepted)
what is an example of a base used in buffer solutions for DNA analysis
what is the pKa of this base
Tris =Tris(hydroxymethyl)aminomethane
8.12
name three potential acids to be used in PCR and why they might be used
acetic acid = good for the use of enzymes but can overheat when used in gels (electrophoresis)
boric acid = inhibits enzymes and less overheating in gels seen with this
hydrochloric acid = no specific use but seen in PCR
what does the water using in buffers need to be
distilled and then filtered so it has a high purity = can be expensive but needed here
can be autoclaved to be ‘nuclease free’
give two other things that are common to be found in buffers and their purpose
EDTA - binds to metal ions that may damage DNA to remove them from solution
salts - e.g sodium, potassium and/or magnesium, can shield the -ve charge on phosphates, help with hybridisation and may be needed for enzyme activity
give two other things that are sometimes used in buffers and their purpose
denaturants - chemicals (e.g urea or formamide) that can help us look at specific areas of DNA as they aid with strand separation
surfactants - examples include SDS or Triton X-100 - help remove unwanted ‘greasy’ elements
