DT - DNA Replication

Question 1

What are 3 methods of DNA replication?

Answer 1

1. Conservative
2. Semi-Conservative
3. Dispersive

Question 2

Describe the Meselson and Stahl experiment

Answer 2

1. Bacteria were grown on an N-15 (heavy) plate
2. Labelled bacteria were then transferred to an N-14 (light) medium
3. DNA is isolated after 1,2,3 generations
4. DNA is separated by density gradient separation

Question 3

What result is seen after each generation?

Answer 3

• At zero: All DNA has N-15
• After 1 generation: All DNA has a density between N-15 and N-14
• After 2 generations: 50% have a density of N-14, 50% is intermediate
• After 3 generations: 75% have a density of N-14, 25% is intermediate

Question 4

What are the 2 main types of density centrifugation techniques?

Answer 4

1. Sucrose density centrifugation
2. Caesium Chloride density gradient centrifugation

Question 5

How does Sucrose density centrifugation work?

Answer 5

• A sucrose concentration gradient already established (more concentrated at the bottom)
• Samples are applied at the top of the tube and eventually everything goes to the bottom
• Non-equilibrium method

Question 6

How does Caesium Chloride density gradient centrifugation work?

Answer 6

• Under the high centrifugal force, the caesium forms a concentration gradient
• Samples disperse and ‘float’ at the position corresponding to their buoyant density
  Equilibrium method
  -

Question 7

How does replication occur in organisms?

Answer 7

Replication starts at a fixed point and is bi-directional
- In eukaryotes, there are multiple replication forks

Question 8

What direction does synthesis occur?

Answer 8

5’ –> 3’

Question 9

What is a Primer made from and what enzyme produces it?

Answer 9

• RNA
• Primase

Question 10

What is made in short fragments from the lagging strand? How are they joined?

Answer 10

Okazaki Fragments
DNA ligase

Question 11

What is DNA sequencing

Answer 11

A general laboratory technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule

Question 12

What modified nucleotide is involved in chain termination?

Answer 12

dideoxynucleotide triphosphate (ddNTP)

Question 13

How are DNA fragments separated?

Answer 13

Gel Electrophoresis

Question 14

How does next-generation sequencing work?

Answer 14

1. Anneal short fragments of DNA to a slide using oligonucleotide adaptors
2. PCR amplified to create multiple copies
3. Anneal 3’-O-blocked reversible terminator nucleotide with fluorescent dye
4. Use of computer algorithms to detect signals and construct a sequence

Question 15

Describe the steps involved in PCR

Answer 15

1. DENATURE DNA to produce single strands
2. Lower temperature to allow primers to anneal
3. Reheat DNA to prevent reforming of H bonds and to ensure optimum enzyme activity
4. Use of Taq DNA polymerase (Can withstand high temperatures)