Workshop 1

Question 1

Why does double stranded DNA form a helical structure rather than an untwisted linear ladder?

Answer 1

• The helical structure brings the bases together.
• This improves pi-pi stacking which is favourable.
• The closing of the gaps excludes water from the hydrophobic environment.
• Helical structure increases the distance between phosphate groups and lowers the electrostatic repulsion.
• The 5’ to 3’ sugars brings the structure together.

Question 2

Would the following changes in condition increase or decrease the melting temperature of a DNA duplex?

Adding NaCl

Answer 2

• Salts are required for stability.
• It will increase the stability therefore increasing the melting temperature.
• The Na+ associates with the helix and screens the negative charges which otherwise impede hybridisation.

Question 3

Would the following changes in condition increase or decrease the melting temperature of a DNA duplex?

Replacing NaCl with the same concentration of MgCl2

Answer 3

• Mg is a bigger molecule so it requires less molecules
• Maintaining the same concentration with a bigger molecule will increase the stability.
• Mg2+ is better at screening charge than Na+ because of its higher charge.
• An increased stability means an increased melting temperature.

Question 4

Would the following changes in condition increase or decrease the melting temperature of a DNA duplex?

Introducing mismatches into the sequence

Answer 4

• Decrease the melting point
• The hydrogen bonding will be mismatched introducing repulsions.

Question 5

Would the following changes in condition increase or decrease the melting temperature of a DNA duplex?

Raising the pH above 10

Answer 5

• Decreases the melting temp
• Hydrogen bond donors will be deprotonated creating mismatch hydrogen bonds so it repells and is less stable and will melt easier

Question 6

Would the following changes in condition increase or decrease the melting temperature of a DNA duplex?

Replacing CG pairs with AT

Answer 6

• Decrease melting temperature
• AT base pairs contribute less to duplex stability because they have less hydrogen bonds.

Question 7

Would the following changes in condition increase or decrease the melting temperature of a DNA duplex?

Making the sequence the longer

Answer 7

• Increase melting temp
• The ends of the duplex are relatively destabilising and increasing the duplex length reduces their significance

Question 8

Considering PCR, why do the two template strands get bound by primers after annealing instead of binding each other?

Answer 8

• ΔG = ΔH - TΔS
• Temperature of annealing is above T_m of DNA but a few degrees lower than T_m of primers, as a consequence binding of a DNA strand to a primer is thermodynamically favoured over forming a duplex.
• Concentration is also an important factor here. Need sufficient primer to drive DNA-primer reaction forward, but not so much that primer dimers are formed.

Question 9

What modification of dNTPs are needed for Sanger sequencing?

Answer 9

• Need to define the stop point to stop polymerisation reaction
• Take OH out so it can’t bridge at 3’ position so it can terminate replication at a specific base.
• dNTPS - remove the OH to make into ddNTPs
• Tagged with fluorophores or radioactive labels in order to enable visualisation of where the termination occurred using gel electrophoresis.

Question 10

What do we need to know to calculate the pH of a buffer solution using a base (e.g. Tris)?

Answer 10

• pH of the buffer solution
• pKa of the buffer compound
• Concentration of the free base
• Concentration of the conjugate acid