Lecture 19 - Genetic engineering

Question 1

What is the overall process of molecular cloning?

Answer 1

Isolate DNA
Cut DNA
Join to recombinant vector
Introduce recombinant vector into bacteria
Amplify recombinant DNA by bacterial growth

Question 2

What is a palindromic sequence?

Answer 2

A sequence that could bind to itself if put in reverse, eg
ACGCGT

Question 3

What is the process of cutting and splicing DNA?

Answer 3

Restriction endonuclease cuts palindromic sequence
The sticky ends can be rejoined

Question 4

What is the detailed process of molecular cloning?

Answer 4

A cut DNA fragment is combined with a cut plasmid using DNA ligase
The cut plasmid contains a selectable marker and origin of replication
The cut DNA fragment and plasmid join and become either:
An unmodified plasmid
A recombinant plasmid
A circularised DNA fragment

Question 5

What is meant by a DNA library?

Answer 5

Involves the isolation and separation of individual sequences within a cell

Question 6

What is a genomic library?

Answer 6

Involves nuclear DNA
All genes

Question 7

What is a cDNA library?

Answer 7

Involves mRNA
Only expressed genes
Cell type specific
Must be converted to DNA to be cloned

Question 8

What is the difference between genomic and cDNA libraries?

Answer 8

c = Comprises expressed genes g = Comprises sequences representing the genome

Question 9

What is the process of obtaining the therapeutic value of proteins?

Answer 9

Fuse coding region of gene to a strong promotor
Insert recombinant gene into host
Host multiplies and overproduces protein
Purify the protein

Question 10

What are the advantages of bacteria as a host system?

Answer 10

Cheap
Grow fast
Easy to maintain
Stable

Question 11

What are the disadvantages of bacteria as a host system?

Answer 11

Proteins might be insoluble or denatured
No post translational modification

Question 12

What are the advantages of animal cells as host systems?

Answer 12

Post translational modification
Soluble / properly folded

Question 13

What are the disadvantages of animal cells as a host system?

Answer 13

Expensive
Unstable

Question 14

What is an example of insulin being created in host systems?

Answer 14

Currently there are only animal sources
This can cause immunological problems and there is potential of cross species infection
This means it might be more suitable to use bacteria to produce human insulin