Basic genetic engineering

Question 1

What is genetic engineering?

Answer 1

Directly manipulating the genetic material of a hoagie, cell or whole organism for a specific purpose

Question 2

What is recombinant DNA technology?

Answer 2

The in vitro (outside a cell) and in vivo (inside a cell) techniques used to manipulate nucleic acids which make genetic engineering possible

Question 3

What end of a DNA strand can nucleotides be added to?

Answer 3

A DNA strand can only be extended by adding a nucleotide to the 3’ OH end

Question 4

What are the steps for gene cloning? (7)

Answer 4

a) extract DNA from cell
b) cut out the gene
c) cut open plasmid
d) insert the gene (ligase)
e) transform recombinant plasmid into a bacterial host and amplify the clone
f) isolate plasmid DNA from a large no. of bacterial cells
g) Remove cloned gene from the plasmid to use as required

Question 5

What are the key tools in recombinant DNA technology?

Answer 5

Restriction endonucleases - cut DNA at specific sites

Question 6

What do bacterial cells use to help protect themselves against foreign DNA entering the cell?

Answer 6

Restriction systems - cut the foreign cell at specific points
Protects its own DNA with restriction and modification enzymes

Question 7

What is a recognition site?

Answer 7

A short sequence of nucleotides in double stranded DNA compromises a specific recognition site

Question 8

How does a bacterium protect its own DNA?

Answer 8

Methylates (A) site : GA(A)TTC

Question 9

How is DNA protected from restriction cutting during DNA replication?

Answer 9

Methylation - restriction enzyme can’t cleave

Question 10

What are the main types of restriction endonucleases and what do they do? (4)

Answer 10

Type 1 - cut at random position
Type 2 - cut within recognition sites
Type 3 - needs 2 recognition sites to cut
Type 4 - only cut DNA with special chemical modifications

Question 11

How do type 2 enzymes cut?

Answer 11

They cut in palindromic sequences - each strand have the same sequence when read in the 5’ to 3’ direction

Question 12

What part of the DNA do type 2 enzymes bind to?

Answer 12

Phosphodiester backbone

Question 13

What re the 2 types of ends that Type 2 enzymes can make on DNA?

Answer 13

Blunt ends

Sticky ends

Question 14

What are the key features of a plasmid? (4)

Answer 14

• Circular with multiple copies per cell
• Carry extra genes for specific circumstances
• Some plasmids can be copied from one bacterial cell to another by bacterial sex
• Only some bacterial cells in a species may possess a certain plasmid at any given time

Question 15

What are the 2 types of plasmids?

Answer 15

• Large (50-100kb), low no. of copies, can transfer cell to cell
• Small (3-10kb), high no. of copies, can’t transfer

Question 16

What do these terms mean:
IS
Tn
Tra operon
OriV
OriT

Answer 16

IS = insertion sequence
Tn = transposon
Tra operon = transfer genes (important)
OriV = plasmid origin of DNA replication
OriT = origin of transfer

Question 17

Why is Tra operon important? (2)

Answer 17

It determines the synthesis of a structure called a sex pilus
This brings 2 cells together and triggers plasmid transfer

Question 18

What is the sex pilus’ role? (4)

Answer 18

• Pilus makes contact and draws the cells together to form a conjugation bridge
• The plasmid DNA is nicked at OriT and rolling circler DNA replication occurs
• The displaced strand is directed into the other cell
• The cells separate each with a plasmid

Question 19

How do Hfr strains form? (3)

Answer 19

• The plasmid integrates into the bacterial chromosome
• This occurs rarely and is generally at sites of homology provided by IS elements
• This can then lead to transfer of chromosomal DNA from one cell to another

Question 20

What is the point of plasmid transfer?

Answer 20

Give the host cell a new function that gives it an advantage of other bacteria

Question 21

Are plasmids smaller or bigger than the chromosome?

Answer 21

Plasmids are smaller

Question 22

Why is conjugation so important for bacteria?

Answer 22

Antibiotic resistance

Question 23

What are they key functions of plasmids as vectors? (4)

Answer 23

• based on small naturally occurring non-conjugative plasmids
• replicate autonomously to high copy number in bacteria
• Carry added antibiotic resistance genes
• carry an added multiple cloning site