Gene Cloning and Fusion Protein Expression

Question 1

How does DNA cloning work?

Answer 1

It yields multiple copies of a DNA segment or gene

Question 2

What features do most cloning methods share?

Answer 2

1. Restriction Enzymes
2. Plasmids
3. Use of Bacteria

Question 3

How do Restriction Enzymes work?

Answer 3

• Recognises specific nucleotide sequences in DNA
• Cuts both strands of the DNA molecule

Question 4

What is a PLASMID?

Answer 4

Molecules of DNA found in bacteria separate from the bacterial chromosome

Question 5

What are the essential features for using plasmids for cloning?

Answer 5

1. ORI
2. Unique restriction site - occurs only once
3. Small size
4. Selectable marker - provides resistance to antibiotic

Question 6

What is a CLONING VECTOR?

Answer 6

A DNA molecule that can carry a foreign DNA into a host cell and replicate there

Question 7

What are the steps to cloning?

Answer 7

1. Vector + DNA
2. Recombinant DNA
3. Transform Host Cells
4. Select for Transformed Cells
5. ID/Screen Host Cells that contain recombinant molecule

Question 8

What is RECOMBINANT DNA?

Answer 8

DNA from two different sources incorporated in vitro into a single molecule

Question 9

What features do EXPRESSION VECTORS have?

Answer 9

1. ORI
2. Unique Restriction Site
3. Small Size
4. Selectable Marker
5. Promoter Sequence
6. RBS (Ribosome Binding Site)
7. TAG Protein to aid purification

Question 10

What is a FUSION PROTEIN?

Answer 10

TAG Protein + Protein of Interest

Results from expression of recombinant DNA