Lecture 23

Question 1

How was the problem of cloning large genomes solved?

Answer 1

Forming clone libraries, made up of small fragments

Question 2

How are clone libraries are formed?

Answer 2

Restriction Enzymes are used to chop the genome to be cloned, the same restriction enzyme is then used to open up the plasmid the chopped up sections are then placed in the vectors through ligases with the vectors being transformed into cells where they can be replicated

Question 3

Where were restriction enzymes originally found and why didn’t they damage the organism they were found in?

Answer 3

In bacteria where they had evolved as a protection mechanism against bacteriophages, they didn’t cut the bacterial genome as the host had methylation patterns which made that DNA inaccessible to the restriction enzyme

Question 4

What occurs when a bacteriophage enters a new host cell?

Answer 4

There is a race between the restriction enzyme and methylation enzyme to reach the invading DNA, normally the restriction enzyme will win this race

Question 5

Why is there a change in phage titre when shifting between bacterial species?

Answer 5

The different bacterial strains have different restriction and methylation sequences, this results in the regions of the phage that are methylated not protecting it from restriction enzymes of the new bacteria so large volumes are destroyed

Question 6

What is unique about the site of restriction enzymes?

Answer 6

They are symmetrical

Question 7

What are the features of acrylamide gel electrophoresis?

Answer 7

Used for one 1bp-1kbp

used for sequencing single stranded DNA

Question 8

What are the features of agarose gel?

Answer 8

0.5-20 kbp

used for plasma cloning

Question 9

What is pulse field gel electrophoresis?

Answer 9

used for very large segments of DNA

50kbp-10Mb

Question 10

What are the different types of restriction enzymes cutters?

Answer 10

4 cutters

6 cutters and 8 cutters

Question 11

What are the average frequencies of cleavage events with each type of cutter?

Answer 11

4 cutters- 1/256
6 cutters- 1/4 kbp
8 cutters- 1/65 kbp

Question 12

How can frequencies of cleavage events for cutters be calculated?

Answer 12

Multiply the frequency of each base in the sequence to determine the frequency of a cleavage base

Question 13

What information needs to be known in order to construct a restriction map?

Answer 13

If the DNA molecule is linear or circular
A estimate of the total size
Estimate of the number of cuts per restriction enzyme
use of a single cutter as an anchor point
Identify the difference in the digests and +/- anchor restriction enzymes
draw possible map and check

Question 14

How can the ends of DNA cut by restriction enzymes be joined together?

Answer 14

Use of a T4 DNA Ligase
Establishes a phosphodiester bond between nucleotides
Requires a free 3’-OH end and 5’-PO4
Can use both blunt and sticky ends
Requires ATP as an energy source and Mg2+ as cofactor

Question 15

What is the role of sticky ends in DNA technology?

Answer 15

The same sticky ends will be generated by and DNA fragment if it is cut by the same enzyme
This means fragments cut by this enzyme can be annealed together
Alternatively blunt ends can be produced through DNA pol 1. allowing greater versatility in combining DNA fragments

Question 16

What do modern cloning techniques use instead of ligase?

Answer 16

Topoisomerase is used as it is faster and more efficient
Site specific recombinases (lambda attachment site to gateway cloning)
Long sticky ends can anneal stably without ligase

Question 17

How can a plasmid be introduced into a bacteria?

Answer 17

Chemical treatments such as Ca2+, Rb2+ and heat shock though this has a size limit of 20 kb
Electroporation apply a square pulse of current to cause pores in wall/membrane
Some bacteria such as Bacillus Subtilis are highly competent

Question 18

What are most plasmid vectors based on and what improvements have been made to these vectors?

Answer 18

Plasmids are based on colE1
there has been an addition of antibiotic resistance for use as a selectable marker
mutations which increase copy number in the cell
removal of undesirable restriction enzyme sites
addition of lac gene to allow a simple blue/white screen
addition of unique cloning sites

Question 19

What are multiple cloning sites?

Answer 19

a series of sites for restriction enzymes placed back to back made through an oligo synthesizer and allows many restriction enzymes fragments to be placed into the same vector