Lecture 19 - Recombinant DNA Technologies Flashcards
What are 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 GFP used for?
Acts as a fluorescent marker
What are critical elements for recombinant DNA technologies?
Plasmids
What are plasmids?
- Circular pieces of dsDNA
- Replicate independently of host’s cell DNA
- Common in bacteria
- Provide a benefit to host e.g antibiotic resistance.
What makes a Eukaryotic cell?
Nucleus
Membrane bound organelles
Often multi cellular organisms
What makes a Prokaryotic cell?
No nucleus
No membrane bound organelles
Uni-cellular
smaller
What are the key components of recombinant DNA plasmids?
- Origin of replication
- Antibiotic resistance gene
- Promoter
- Selectable marker
- Restriction sites
Why are the origin of replications needed?
Allows for initiation of replication using host DNA polymerase.
Why are Antibiotic resistance genes needed?
Provides survival advantage to cells containing plasmid
Why are Promoters needed?
Drives expression of your favourite gene in cells with appropriate transcription factor machinery
Why are selectable markers needed?
To select for cells that have successfully taken up the plasmid. e.g the GFP gene.
Why are restriction sites needed?
Allows ligation of gene of interest into the cloning vector
Promoters are?
Highly specific - need different promoter across species even if for same gene.
What are restriction enzymes?
Proteins isolated from bacteria that cut DNA
- cuts dsDNA at specific sequences.
How is DNA inserted into a plasma?
Restriction enzymes cut specific regions of DNA,
DNA is inserted in gap
What joins the DNA strands together after being cut and DNA inserted?
DNA ligase - via phosphodiester bonds
What is the name for a plasmid after a viral gene is added?
Vector
What is transformation?
Transfer of vectors into bacteria
What are the steps of transformation?
- Transfer of vector into bacteria
- Leave for time to grow and divide with recombinant vectors
- Only cells that take up the plasmid with survive creating a pure sample (due to antibiotic resistance gene)
What does it mean by universal genetic code?
All organisms read the same codons as the same amino acids
What is the significance of the universal genetic code?
Can transfer genes from bacterial cell into human cell, and vice versa.
What do eukaryotic genes have that prokaryotic genes do not?
Exons and introns.
What would introns in prokaryotic genes cause?
Would lead to a nonsense polypeptide that did not fold into correct tertiary structure
What allows for prokaryotic Pre-mRNA to turn into working mRNA
Removal of introns
What is cDNA?
Coding sequence DNA - Eukaryotic DNA with introns removed
What is the process of mRNA transcribing back into DNA called?
Reverse transcription
What enzyme carries out reverse transcription?
Reverse transcriptase
Why is cDNA useful?
No introns allows successful translation to a functional protein in prokaryotic cells.
Without introns in sequence, the overall size of the insert is reduced.
