Vectors

Question 1

How are restriction sites incorporated in primers?

Answer 1

Within 5’ tag with a few extra bases for endonuclease

Question 2

How can restriction digests provide orientation for gene insertion?

Answer 2

Different forward and reverse sites

Question 3

What action do alkaline phosphatases have?

Answer 3

Remove 5’ phosphate from vector to prevent religation

Question 4

When are alkaline phosphatases inactivated?

Answer 4

When the gene insert is added

Question 5

What type of DNA do restriction enzymes target?

Answer 5

Unmethylated- host must be methylation disabled

Question 6

What types of sequence do type 2 restriction enzymes digest?

Answer 6

pallindromic to leave sticky or blunt

Question 7

What is an alternative method to insert genes?

Answer 7

Transposition

Question 8

What size are natural plamids?

Answer 8

1-100kb

Question 9

How many natural copies of plamids do cells have?

Answer 9

Up to 1000

Question 10

What size insert do plamids carry?

Answer 10

10kb

Question 11

How many copies of a plasmid are transformed into a cell?

Answer 11

15-20 for natural levels

Question 12

Why are synthetic plasmids used in cloning?

Answer 12

They replicate independently and no not undergo horizontal gene transfer

Question 13

What characteristics do plasmids have?

Answer 13

OriC, MCS, drug resistance markers

Question 14

How can cells be transformed?

Answer 14

Electroporation, CaCl2 and heat shock, CaPO3 precipitation, liposomes or mechanical bombardment

Question 15

How can transformation be observed?

Answer 15

Antibiotic resistance, blue/white selection, colony PCR, hybridisation and restriction digest

Question 16

What are key characteristics of host cells?

Answer 16

Restriction enzyme deficient
No mutation or rearrangement of DNA
Auxotrophic

Question 17

What is the structure of bacteriophage lamda?

Answer 17

dsDNA linear genome of 49kb with 12bp cohesive ends separated by 37-40bp
Viral packaging

Question 18

How do bacteriophages insert DNA into a cell?

Answer 18

Transfection

Question 19

What are is the lytic cycle of phage lamda?

Answer 19

Transfection,
Hijacks host machinery for rolling circular replication
Catenane is cleaved at cos sites and associates with preformed packaging heads and tails
Lysis of cell membrane

Question 20

What is the lysogenic lamda phage cycle?

Answer 20

Genomic integration forms prophage
Repressor molecule for lytic cycle
Turbid plaques produced

Question 21

Which type of lamda cycle produces phages resistant to reinfection?

Answer 21

Lysogens

Question 22

What is the lamda packaging constraint?

Answer 22

78-105%

Question 23

What are the 2 types of gene insertion into lamda vectors?

Answer 23

Replacement with 10-22kb stuffer fragment

Insertion of up to 1-10kb fragment

Question 24

Which type of non essential DNA is removed from phages?

Answer 24

Between capsid and DNA synthesis genes, involved in lysogeny

Question 25

What are cosmids?

Answer 25

Plasmids that contain a cos site for transfection as phage

Question 26

What features does the cosmid genome contain?

Answer 26

``` OriC Cos Insert up to 40kb Restriction site Antibiotic resistance ```

Question 27

What features are replaced by the insert in a cosmid?

Answer 27

Packaging and lysis genes

Question 28

What size is the M13 phage?

Answer 28

6.4kb ssDNA genome with no packaging constraint

Question 29

What size inserts does M13 accept?

Answer 29

10kb due to rearrangement

Question 30

What is the M13 life cycle?

Answer 30

Targets male specific/ F pilus Replicates at dsDNA Produces ssDNA for release No cell death

Question 31

What is M13 used for?

Answer 31

Was used for sequencing but now used for phage display with gene3 replaced for expression of recombinant protein on capsid surface

Question 32

What type of cells is mechanical bombardment used to transform?

Answer 32

Plant cells through cell wall

Question 33

How efficient is liposome transformation?

Answer 33

90%

Question 34

Which cells is Electroporation used for?

Answer 34

Native animal, treated yeast cells

Question 35

How does calcium phosphate precipitation transform cell?

Answer 35

Endocytosis with 30-50% efficiency