1
Q
STEP ONE - How would you obtain the required gene? - Using mRNA
A
- Obtain the mRNA for the gene from the cell where that gene is being expressed. e.g. human insulin obtained from the pancreas
- to the mRNA, add some DNA nucleotides and the enzyme reverse transcriptase
- this enzyme catalyses the opposite reaction to transcription
- allows for the conversion of mRNA into single strand of DNA (cDNA)
- DNA is made double stranded by adding DNA nucleotides, primers and DNA polymerase
two copies of the gene are made
2
Q
STEP ONE - How would you obtain the required gene? - automated machine
A
- Gene can be synthesised in a lab using an automated machine by adding DNA nucleotides, DNA polymerase and provide a sustainable temperature
3
Q
STEP TWO - what happens when 2 sections have been cut off?
A
- When 2 sections of DNA have been cut off with the same restriction enzyme, giving them complementary sticky ends, they can be joined together using DNA ligase
- when the pieces join, it is called Recombinant DNA
4
Q
What are marker genes?
A
- Chosen gene is inserted into a plasmid, one marker gene is also inserted at the same time
5
Q
STEPS 3 - What are vector areas?
A
- Vectors area is added to the solution containing the chosen host cell
- DNA does not easily cross the cell membranes
6
Q
STEP 3 - What is Heat shock treatment?
A
- This makes the walls and the mebrane more porous allowing for the uptake or recombinant vector?
7
Q
STEP 3 - What is electoporation?
A
High voltage pulse applied to a host cell to disrupt the cell surface membrane
8
Q
STEP 3 - What is electtrofusion?
A
- Electrical fields are used to introduce DNA into the host cells
9
Q
STEP 4 - Hint: host cells
A
Indentifying which host cells have been successful int the taking up the new gene
