Stability and Handling

Question 1

What do you need to calculate to know the equilibrium of the DNA duplex stability?

Answer 1

ΔG

Question 2

What is the ΔG equation?

Answer 2

ΔG = ΔH - TΔS
Where ΔH = change in enthalpy (heat), T = temperature, ΔS = change in disorder/entropy
* Delta G must be negative to be stable, the more negative the more stable

Question 3

What are some factors that affect the stability of the double helix?

Answer 3

• Temperature
• Salt concentration
• pH
• Neighbouring bases
• Ion charge

Question 4

How does neighbouring bases affect the stability of the double helix?

Answer 4

• Due to π-stacking
• GC has more hydrogen bonds so it has a larger delta G, therefore more stability and increase in melting temperature

Question 5

Why is DNA/double helix more stable that RNA/single strands?

Answer 5

• Double helix = more ordered and therefore more entropic penalty, H-Bonds + π-stacking means enthalpic gain
• Single strand = less ordered and therefore entropic gain, H-bonds broken and therefore enthalpic penalty

Question 6

How do you measure duplex stability?

Answer 6

Add all the delta G’s together for each bond
* All calculations are performed in 1M NaCl

Question 7

Duplex stability in response to temperature

Answer 7

• Delta G will increase as temp increases and DNA will become unstable
• Increasing conc of delta S as temp increases

Question 8

Duplex stability in response to salt conc

Answer 8

• DNA needs cations to shield the charge of the phosphate groups
• Increasing salt conc pushes up the melting temperature - increased stability
• 2+ cations are more strongly stabilising than 1+ cations - less ions required to make DNA more stable

Question 9

Duplex stability in response to pH

Answer 9

• Reduced pH = protonation occurs, hydrogen bonds are borken, protonate our amines which then detabilises our DNA, if pH is low enough you can completely change the chemistry
• Increased pH = deprotonated occurs in hydrogen bonds, creating HB mismatches whihc will be repulsive, degradation, decrease in melting temperature,

Question 10

How does introducing mismatches into the sequence effect the melting temperature of the DNA duplex?

Answer 10

Decrease, H bonds will be mismatched introducing repulsions

Question 11

How would making the sequence longer effect the melting temperature of the DNA duplex?

Answer 11

Increase, the ends of the duplex are relatively destabilising and increasing the duplex length reduces their significance

Question 12

What are the main 2 thigns in a buffer and why is it used?

Answer 12

Mixture of a base and it’s conjugate acid and used to maintain a particular pH
* Base: Tris - Tris(hydroxymethyl)aminomethane
* Acid: Acetic acid (good for use of enzymes, can overheat in gels), Boric acid (inhibits enzymes, less overheating in gels), HCl (used in PCR)

Question 13

Common and uncommon things

What else in in buffers?

Answer 13

Common:
* EDTA = binds stray metal ions which could damage DNA
* Salts = sodium, potassium and/or magnesium, as a need for hybridisation
Sometimes:
* Denaturants = urea or formamide, ensures bands on gels represent linear single strands
* Surfactants = mop up unwanted greasy matter (cellular material)

Question 14

What is the equation called that is used to make a buffer/

Answer 14

Henderson-Hasselbalch

Question 15

What is the Henderson-Hasselbalch equation?

Answer 15

pH = pKa + log10([B]/[BH+])
* pH of the buffer solution
* pKa = of the buffer compound (i.e. Tris)
* [B] = concentration of free base
* [BH+] = concentration of conjugate acid

Question 16

Make a pH 8 Tris/acetate buffer at a concentration of 45 mM, pka = 8.12
(i.e. How much acid do we need to add ([BH+]) to generate a pH 8 Tris/acetate buffer at 45mM?)

Answer 16

8 = 8.12 + log10([B]/[BH+])
[b] + [BH+] = 0.045 –> [B] = 0.045 - [BH+]
-0.12 = log10([B]/[BH+])
[B]/[BH+] = 0.759
(0.045 - [BH+])/[BH+] = 0.759
[BH+] = 0.0255 M

Question 17

How do you measure the concentration of DNA?

Answer 17

Beer-Lambert Law: A = ε c l
* ε = absorpivity in M-1cm-1 (constant)
* c = concentration
* l = path length in cm
* A = Absorbance

Question 18

What is ε for DNA?

Answer 18

at wavelength = 260 nm
dsDNA = 0.020 (μg/ml)−1cm−1
ssDNA = 0.027 (μg/ml)−1cm−1
RNA = 0.025 (μg/ml)−1cm−1

Question 19

What instruments are used to measure the conc of DNA?

Answer 19

• UV-Vis Spectrophotometer = Requires 1-3mL, One sample at a time, Collects whole spectrum
• UV-Vis Plate Reader = Requires 50-300uL, Many samples as once, Whole spectrum or single wavelength
• NanoDrop = Requires 1uL, One sample at a time, Usually single wavelength