Eukaryotic genetics and recombinant DNA technology

Question 1

Why do eukaryotes need chromatin when prokaryotes don’t?

Answer 1

• Eukaryotes have much larger genomes which need to be tightly packaged to fit into the nucleus.
• DNA must be compacted into the nucleus to allow it to be transported into new cells during cell division.

Question 2

How do histone proteins bind to DNA in chromatin?

Answer 2

• DNA backbone contains many negatively charged phosphates.
• Histone proteins are positively charged.

Question 3

What are 2 processes of control of the packaging of chromatin?

Answer 3

• Chromatin Remodelling Enzymes use energy from ATP hydrolysis to remodel chromatin affecting packing of chromatin.
• Post-translational modifications of histone tails: can be acetylated, methylated or phosphorylated. Can lead to more open or condensed chromatin.

Question 4

What are the key differences between eukaryotic and prokaryotic DNA replication?

Answer 4

In Eukaryotes:
* Only occurs during S phase of cell cycle.
* Larger genome, slower DNA polymerase (chromatin forms obstacle), multiple origins of replication required.
* Differences in end of replication as chromosomes are linear not circular.

Question 5

How does transcription in eukaryotes differ from that in prokaryotes?

Answer 5

• Transcription occurs in the nucleus, different location to translation.
• There are multiple RNA polymerases in eukaryotes, only 1 in prokaryotes.
• Eukaryotic RNA polymerases require many more accessory proteins in order to function.
• Promoters are more complex

Question 6

What are the four types of RNA polymerases and what type of RNA do they synthesise?

Answer 6

• RNA Polymerase I: rRNA
• RNA Polymerase II: mRNA
• RNA Polymerase III: tRNA
• Mitochondrial RNA Polymerase

Question 7

What is mRNA processing and what are the 3 main types?

Answer 7

• Pre-mRNA becomes Mature-mRNA.
• Does not occur in prokaryotes.
• Occurs during transcription.
  1. 5’ Capping
  2. mRNA splicing
  3. Polyadenylation

Question 8

What is the purpose of 5’ Capping and polyadenylation?

Answer 8

Protect mRNA from degradation by exonucleases. Increase mRNA stability.

Question 9

Why does eukaryotic mRNA contain introns and exons?

Answer 9

Allows many different proteins to be made from the same gene (different combinations of exons to form the protein).

Question 10

How is DNA prepared?

Answer 10

1. Cells are homogenised (mechanical disruption of cells). There are other methods for breaking cells to extract DNA but this is the most common.
2. Transfer the supernatant (aqueous clear phase) to new test tube, add phenol and centrifuge again. This removes the protein: aqueous layer contains DNA and RNA.
3. Transfer the aqueous layer to a new tube and add ethanol and high salt. Causes nucleic acid to precipitate.
4. Centrifuge and keep precipitate, removing supernatant above.

Question 11

What are the main properties of Type II Restriction Endonucleases and what do they do?

Answer 11

• Sequence specific
• Often palindromic
• 4 to 8 base pairs long
• Cleave both strands of dsDNA.
• Often a straggered cut (sticky ends).

Question 12

How often do restriction endonucleases cleave?

Answer 12

number of base (4) raised to the power of the length of the recognition sequence.
If length = 6, enzymes cuts once every 4096bp.
Assumes random and balanced distribution of bases.

Question 13

What is the role of DNA ligase?

Answer 13

Covalent bonding of fragments together, forming sugar-phosphate backbone.

Question 14

What is agarose gel electrophoresis and what are its uses?

Answer 14

Process that separates DNA fragments based on size and charge.
Agarose is a polysaccharide (gel material).
Can be used to purify DNA, understand and compare restriction endonucleases.

Question 15

What is recombinant DNA?

Answer 15

An artificial DNA sequence not found in nature: generated by combining DNA from multiple organisms.

Question 16

What is gene cloning

Answer 16

The ability to isolate and make identical copies of a fragment of DNA, such as a single gene.

Question 17

Outline the process of gene cloning.

Answer 17

1. Isolate bulk DNA.
2. Use restriction endonuclease enzymes to isolate a specific sequence of DNA.
3. Ligation of DNA fragment with bacterial plasmid.
4. Force bacteria to take up vector containing DNA insert.
5. Screen the bacteria and grow a colony containing recombinant plasmid clone.

Question 18

What 3 things must all plasmid vectors contain?

Answer 18

• Origin of replication.
• Antibiotic resistance gene not originally in bacterial genome (can be used as a selectable marker: cells which survive exposure to antibiotic must contain viral vector).
• Sites for restriction endonuclease enzymes to produce sticky end complementary to DNA fragment to be cloned.

Question 19

How would a plasmid containing recmobinant DNA be introduced into bacteria?

Answer 19

• Treat with calcium chloride.
• Heat pulse at 42 degrees C. (causes cell membranes to become more porous).

Question 20

How can we verify clones (ensure that plasmid has not reformed without gene)?

Answer 20

Purify plasmid out, cut with restriction endonucleases and use gel electrophoresis.

Question 21

How do we clone eukaryotic genes whilst ensuring that introns are not present?

Answer 21

• Use reverse transcriptase to synthesise complementary DNA from mRNA.
• We can use synthetic T-primer to start this process as all mRNA has poly-A tail.
• RNA is then digested from the DNA hybrid, hairpin forms that acts as a primer for DNA polymerase to reform complementary DNA strand to replace RNA.

Question 22

3 keys steps and explanation of 3rd step.

Describe the PCR process.

Answer 22

1. Template dsDNA is heated to 95 degrees C, denaturing all H-bonds to generate 2 ssDNA templates.
2. Reaction cooled down to annealing temperature for primers (roughly 60 degrees C).
3. Reaction is heated to 72 degrees C: optimal temperature for activity of Taq DNA Polymerase.
4. Primer does not dissociates because DNA Polymerase rapidly extends the primer, stabilising association.
5. Cooled at end to store reaction.

Question 23

How can we measure primer binding strength (a key variable in PCR)?

Answer 23

t_m is the temperature at which 50% of the primer is annealed to the template.
- Affected by length (longer = more H-bonds = stronger).
- Affected by sequence (more CGs = more hydrogen bonds).

Question 24

What are the 3 key uses of PCR?

Answer 24

• Gene Cloning that is not dependent on positions of restriction endonuclease sites.
• Viral Screening e.g. COVID test - amplification allows us to detect virus. Would have to use reverse transcriptase rather than DNA polymerase as desired sequence is RNA - needs to be turned into ssDNA.
• Forensics never enough DNA left at crime scene - needs amplification for analysis.

Question 25

Briefly outline the Sanger Sequencing method.

Answer 25

• Nucleotides made without 3’ OH group (chain terminating ddNTPs).
• Small amount of each of the 4 ddNTPs added, each tagged with different fluorophore.
• Resulting small fragments of different lengths.
• Gel electrophoresis allows us to view different lengths.
• When combined with fluoresence, we can read off sequence.

Question 26

What are the 2 key applications of Sanger sequencing?

Answer 26

• Screen for gene mutations and sequence variants.
• Can validate sequence of PCR product (required as Taq polymerase is more error prone).