Gene Cloning 2

Question 1

How are the vector and DNA fragment combined?

Answer 1

Foreign DNA is digested with a restriction enzyme and the bacterial plasmid is then cut with the same restriction enzyme.
Different DNA sequences cut with the same enzyme can join together as the enzyme makes staggered cuts creating specific sequences. These specific sequences are called sticky ends and can anneal together using DNA ligase.

Question 2

How is the DNA isolated from the cell?

Answer 2

Lyse cell by chemical & physical methods e.g. sonication, homogenization
Remove membrane lipids with detergent (SDS)
Remove proteins by adding a protease
Remove RNA with RNase
Precipitate DNA with alcohol

Question 3

What does DNA ligase do and what does it require for action?

Answer 3

Joins DNA strands together by catalysing the formation of a phosphodiester bond.
Requires ATP.

Question 4

What type of cloning is it when only one restriction enzyme is used?

Answer 4

Non directional cloning

Question 5

What is the problem with Non-directional cloning? and how can the problem be prevented?

Answer 5

The plasmid will often self ligated where its sticky ends match.
The vector needs to be dephosphorylated to minimise self-ligation - using alkaline phosphatase.

Question 6

What type of cloning is it when two restriction enzymes are used?

Answer 6

Directional cloning.

Question 7

What type of cloning is blunt end ligation?

Answer 7

Non-directional cloning (as there is no sticky ends).

Blunt end ligation occurs 100X slower than sticky-end ligation.

Question 8

How do you extract DNA fragments from agarose gels?

Answer 8

Cutting out target bands from gels
Melting agarose gels at around 50°C, the of melting point  of agarose have been lowered by NaI.
Purification of the DNA fragments
- Glass beads
- Spin column-based method

Question 9

What is an alternative strategy of DNA cloning?

Answer 9

PCR-based cloning

Question 10

What is a primer?

Answer 10

Primers are oligonucleotides complementary to different regions on the 2 strands of DNA template (flanking the region to be amplified).

Question 11

How large are primers?

Answer 11

~15-20bp

Question 12

How do the primers bind to DNA in relation to each other?

Answer 12

One hybridises to one strand of dsDNA and the other hybridising to the other strand, such that both primers are oriented with their 3’ ends pointing towards each other.

Question 13

What happens after the primers have hybridised to dsDNA?

Answer 13

The primer is extended from its 3’ end by DNA polymerase.

Question 14

What is the annealing temperature for primers? And how is the temperature linked to primer size?

Answer 14

~55°C

Longer primers require higher annealing temperatures than short primers.

Question 15

Why is Pfu polymerase used instead of Taq polymerase in PCR based cloning?

Answer 15

It has a higher fidelity due to its 3’ to 5’ exonuclease proof reading activity.

Question 16

How can PCR products that were amplified by Pfu polymerase be used for TA cloning?

Answer 16

Addition of A overhangs to the blunt PCR product, using Taq polymerase and dATP.

Question 17

What is TOPO cloning?

Answer 17

Vectors are covalenty bound to topoisomerase I which catalyses the ligation of the 2 ends in 5 minutes at room temperature.

Question 18

What is TA cloning?

Answer 18

Taq polymerase adds an A overhang to the 3’ end of the product- always get a sticky end and non-directional cloning, as both ends of the vector has a T overhang for the A to pase pair with.