genetic engineering

Question 1

What are restriction enzymes?

Answer 1

They are a special class of naturally occurring proteins. Restriction endonucleases breaks the phosphodiester bond on each of the DNA strands at a specific sequence of bases known as the restriction site.

Question 2

What are the 4 steps in DNA cloning?

Answer 2

1. Gene isolation
2. Gene splicing (restriction digestion + ligation)
3. Bacterial transformation
4. Selection (+ confirmation)

Question 3

Sample answer for “describe the steps that will be adopted to clone the DNA insert into ___”

Answer 3

1. Genomic DNA is isolated from (jellyfish).
2. Restriction digestion is performed on (PBR322) plasmid vector and (GFP) insert using (Ptsl). DNA ligation is performed to ligate both digested (PBR322) plasmid and digested (GFP) insert using DNA ligase to form the recombinant plasmid.
3. Recombinant plasmid is transformed into (tetracycline)-sensitive bacterial host cells made competent by calcium chloride treatment with heat shock. Selection is performed by plating the transformed bacteria on selection medium containing (tetracycline).
4. Colonies which grow on the selection medium and (fluorescence a brilliant green when viewed with a hand-held UV light) are likely to be transformed bacteria containing recombinant plasmid because of the expression of the (Tc^R) gene that confers resistance to (tetracycline) and the expression of the (GFP) gene that (fluoresce green under UV light).

Question 4

What are some properties of bacterial plasmids?

Answer 4

• Low molecular weight: more resistant to damage during plasmid extraction / manipulation
• Presence of at least 1 selectable marker: either a form of resistance to antibiotics, a visible colour/fluorescence or cytotoxicity
• Presence of a MCS: region on plasmid that is where gene-of-interest/DNA is inserted

Question 5

What are the two ways in which selection + confirmation is carried out? What is the similarity?

Answer 5

Restriction digestion, or polymerase chain reaction (PCR). Both still need agarose gel electrophoresis to visualise the fragments.

Question 6

Preliminary confirmation using restriction digestion and PCR?

Answer 6

Restriction digestion: Non-recombinant plasmids yield a single DNA band corresponding in size to the cloning vector DNA. Recombinant plasmids will yield 2 bands, 1 corresponding to the vector DNA and the other to the insert DNA.
PCR: Non-recombinant plasmids will yield a small product corresponding in size to the MCS region between the primer binding sites. Recombinant plasmids will yield a larger product consisting of the insert and residual MCS sequences.

Question 7

Genetic engineering is…

Answer 7

Genetic engineering is a process used to modify the genetic material of an organism.

Question 8

A genetically-modified organism is…

Answer 8

A genetically-modified organism is an organism that has its DNA modified by artificial means.