Molecular Genetics (Rose Murray 1-4)

Question 1

Whose experiments concluded that DNA is found inside cells and not proteins?

Answer 1

Hersey and Chase (1952)

Question 2

How did Rosalind Franklin (& Raymond Gosling) help Watson and Crick make their famous discovery?

Answer 2

produced the first x-ray diffraction image of DNA

Question 3

Name the Pyrimidines

Answer 3

Thymine, Cytosine and Uracil

Question 4

Name the Purines

Answer 4

Guanine and Adenine

Question 5

How does alternative splicing provide phenotypic variation?

Answer 5

some introns may be left, coding for a different protein from the same gene.

Question 6

3 main differences between eukaryotic and prokaryotic translation?

Answer 6

• Eukaryotic transcripts leave the nucleus before translation, whereas in prokaryotes, translation occurs right away.
• Eukaryotic transcripts have a polyA tail and a 5’ cap, whereas prokaryotes have neither.
• Single gene per eukaryotic mRNA transcript, however, prokaryotic transcripts can be polycistronic.

Question 7

What is eDNA?

Answer 7

Environmental DNA, found in soil, water, faeces, air etc

Question 8

3 points about prokaryotic DNA packaging

Answer 8

• Arranged in nucleoids (not membrane-bound organelles)
• Supercoiled around architectural proteins (not histones)
• Singular, circular DNA with non-essential genes on plasmids in cytoplasm

Question 9

3 points about eukaryotic DNA packaging

Answer 9

• Packaged into chromosomes
• Supercondensed around histones
• located in the nucleus

Question 10

What is significant about the inheritance of chloroplast DNA (cpDNA)?

Answer 10

The type of egg cell determines the type of chloroplast.

Experiment: Mirabilis plants
eggs cell determined whether chloroplasts were white, green or both (variegated). Sperm had no effect.

Question 11

Which plasmid can the genes for conjugation be found?

Answer 11

F plasmid

it can pop in and out of the chromosome by homologous recombination.

Question 12

What is the role of Topoisomerase in the initiation of DNA replication?

Answer 12

relieves the tension of wound up DNA by breaking and rejoining it ahead of the replication fork.

Question 13

What is the role of Helicase in the initiation of DNA replication?

Answer 13

Unwinds and separates the DNA strands

Question 14

What is the role of Primase in the initiation of DNA replication?

Answer 14

makes RNA primers by using the DNA as a template.

Question 15

What is the role of DNA polymerase III in the initiation of DNA replication?

Answer 15

synthesises new DNA from the 5’ to 3’ direction.

Question 16

What is the role of DNA polymerase I in the initiation of DNA replication?

Answer 16

on the lagging strand, removes the RNA primers and synthesises DNA fragments in its place.

Question 17

What is the role of DNA ligase in the initiation of DNA replication?

Answer 17

joining of the fragments

Question 18

In DNA extraction, what is the role of detergents?

Answer 18

To break up fats/lipids so the membrane opens.

Question 19

In DNA extraction, what is the role of salts?

Answer 19

to weaken the H bonds between DNA strands.

Question 20

In DNA extraction, what is the role of EDTA?

Answer 20

grabs ions with a valency of 2 (can make 2 bonds) and prevents DNAses from working.

Question 21

In DNA extraction, what is the role of Tris?

Answer 21

a buffer to stabilise an alkaline pH.

Question 22

In DNA extraction, what is the role of isopropenol or ethanol?

Answer 22

DNA is insoluble in them so it precipitates out.
It can then be caught by a silica matric in via a spin column.
Ethanol also used to wash out salts.

Question 23

In synthetic oligonucleotide synthesis, what are used insted of dNTPs?

Answer 23

Phosphoamidites

they’re sequentially added to the column to build up a chosen sequence via a coupling reaction. The chain is grown from 3’ to 5’, opposite to normal.

Question 24

What are the ingredients of a PCR mix?

Answer 24

dNTPs, Primers, Buffer, DNA template and Taq polymerase

Question 25

What are the three stages of PCR and their respective optimum temperatures?

Answer 25

Denature strands - 94°C Annealing of primers - 60 °C (depends on the primer) Extension -72 °C

Question 26

How can PCR be used to enable easy cloning into vectors?

Answer 26

Add restriction sites to the ends of the PCR product

Question 27

What two antibiotics are most bacterial vectors resistant to?

Answer 27

Ampicillin and Kanamycin

Question 28

What does the LacZ gene code for?

Answer 28

beta-galactosidase enzyme

Question 29

How does lactose regulate the Lac operon?

Answer 29

The Lac I gene is always switched on, producing a repressor molecule which binds to the regulatory region, preventing RNA polymerase from transcribing the rest of the operon. If lactose is present, it binds to the repressor, inhibiting it and the Lac operon is expressed, producing beta-galactosidase.

Question 30

How can we use the LacZ gene to screen for recombinant bacteria?

Answer 30

we can culture the colonies on IPTG or X-gal, which replaces the function of lactose in the pathway. Wild-type, non-recombinant bacteria will produce beta-galactosidase and X-gal will be broken down into galactose and a blue colour. The LacZ∆M15 mutants (recombinants) will have taken up the DNA, interrupting the LacZ gene, preventing its translation. As a result, these colonies will grow white and can be selected.

Question 31

In sequencing, what is the difference between dNTPs and ddNTPs?

Answer 31

dNTPs have only lost one OH group so can still make connections with other nucleotides. ddNTPs have lost both OH groups so can no longer make connections and terminates the chain.

Question 32

Briefly, explain Sanger Sequencing

Answer 32

PCR is carried out with dNTPs and ddNTPs (e.g. ddGTP) everytime a ddNTP is incorporated, the chain terminates. This produces different lengths of DNA which can be analysed by gel electrophoresis. The ddNTPs can also be fluorescently probed with different colours and automated process can produce a chromatograph.