B: Recombinant DNA

Question 1

• What is a cDNA library?

Answer 1

mRNA is converted into DNA using reverse transcriptase –> called cDNA (complementary DNA)

Question 2

-What is DNA ligase - how does it work?

Answer 2

DNA ligase joins DNA molecules by ligating nicks in double stranded DNA.
DNA ligase is very specific and can only join 2 DNA molecules together when 3’OH of one molecule is immediately adjacent to ‘5 phosphate of the other molecule.

Question 3

-What is a restriction enzyme + explain how EcoRI cuts DNA?

Answer 3

Restriction enzymes used to cleave foreign DNA for protection.
Bacteria methylate their own DNA to make it immune to cleavage by their own enzyme.
EcoRI cuts double stranded DNA at GAATC.

Question 4

–Explain how two DNA fragments can be cut at a specific sequence and joined together.

Answer 4

Restriction enzymes cut DNA at specific sequences, and creating overhanging sites.
Overhanging sites are complementary to each other so anneal and are sealed together by DNA ligase.

Question 5

—What is a plasmid? What are the essential features for a plasmid?

Answer 5

Features include
- E.Coli promoter
- Origin of replication
- Antibiotic resistance gene
- Restriction sites for restriction enzymes

Question 6

—Explain how a piece of human DNA can be inserted into a plasmid.

Answer 6

• Restriction enzymes are used to cut DNA and plasmid samples.
• Cut DNAs are incubated together in DNA ligase so they are joined covalently
• Grow bacteria in presence of selectable marker for plasmid
• Many plasmids taken up by bacteria will have inserts of human DNA at restriction sites.

Question 7

—Explain how these are used:
1 - restriction enzymes
2 - DNA ligase
3 - reverse transcriptase
4 - plasmids
5 - strong promoter

Answer 7

1 - For cutting DNA
2 - Glueing DNA
3 - Converting mRNA into cDNA so that introns can be removed
4 - Used as vehicles for carrying inserted foreign DNA
5 - For expressing foreign DNA in host (e.g. E.Coli promoter)

Question 8

— Explain how insulin can be overproduced in bacteria using DNA recombinant technology.

Answer 8

1. Insert E.Coli promoter upstream of human insulin gene
2. Insert gene and promoter into plasmid
3. Transform plasmid into E.Coli
4. E.Coli produces mRNA from human gene and translates it into protein