Recombinant DNA technology Flashcards
What is the definition of recombinant DNA technologies?
Joining bits of DNA together (sometimes from different species). These are then inserted into an organism to produce (express) a useful protein.
What is an example of a gene used in recombinant DNA technologies?
Green fluorescent protein (GFP) (from jellyfish)
Describe the molecular structure and features of a plasmid (3)
- Derived from endogenous DNA
- Circular, double stranded
- Replicate independently from host’s chromosomal
What are 4 key components of a plasmid?
- Origin of replication (ORI)
- Antibiotic resistance gene
- Promoter
- Restriction site
What is the role of the origin of replication?
allows initiation of replication using host DNA polymerase
What is the role of the antibiotic resistance gene?
allows selection of the cells which have take up the protein/plasmid (all others killed by antibiotic)
What is the role of the promotor?
Drives expression of your favourite gene (e.g. insulin or GFP) in cells with appropriate transcription factor machinery
What is the role of the restriction site?
so that you can put what you want into the backbone
What are 2 tools used for recombinant DNA technologies?
- Cutting and pasting DNA into plasmids
- Amplifying plasmids - transformation
What 2 things are used for cutting and pasting DNA into plasmids?
restriction enzymes and DNA ligase
What is the general function of restriction enzymes and where are they naturally found?
○ Naturally found in bacteria
○ Used to stop invading pathogens/phages entering the cells
= defence system to degrade foreign DNA
What do restriction enzymes do in terms of recombinant DNA technologies?
Cuts dsDNA at specific sequences
- Has to be double stranded and unmethylated (= from hosts)
- Cuts so that there’s overhang = sticky ends
What are sticky ends used for?
Add to what we want to insert to plasmid with corresponding sticky ends, then repair DNA
What does DNA ligase do in terms of recombinant DNA technologies?
Catalyses the formation of phosphodiester bond to repair nick in DNA backbone
What is transformation?
transfer of plasmids into bacteria
Which bacteria do we transform?
the bacteria selected by antibiotic resistance contained on plasmid
What is the difference between using a plasmid with a bacterial gene vs with a eukaryotic gene when inserted into bacteria?
The gene will be expressed if bacterial, only replicated lots if eukaryotic
What is the difference between prokaryotic and eukaryotic genes, and what does this mean for prokaryotes?
Prokaryotic genes don’t have introns, and thus prokaryotes don’t have the machinery to process eukaryotic introns
What is the significance of having a universal genetic code?
• All organisms read the same codons as the same amino acids
.: we can transform a human gene into bacteria and it will still make the same protein
To use prokaryotes to replicate a eukaryotic gene, which part(s) of the gene are used?
coding sequence only!
When and why is it best to transform the plasmid into bacteria?
- Bacteria are best if don’t need post-translational modification/folding
- Fast and cost-effective
If the protein of interest requires post-translational modification, what are the best cells to use?
Mammalian, transgenic
what are examples of post-translational modification? (2)
cleaving C chain, glycosylation
What cells are used to make EPO? Why?
CHO cells - very good at glycosylating proteins
What protein is goat milk used to produced? What is its function?
anti-thrombin (AT) - Blocks the function of thrombin, which is in the clotting cascade
How is AT produced?
milk specific promotor added to human AT gene (is a mammary cell specific protein so AT not produced in every cell for whole life). AT is then produced in the milk and purified out
