L19: Recombinant DNA Technology Flashcards
Why was treatment in the 1950s for diabetes (DNA sequencing) not always 100% pure?
Because insulin from cattle and pigs were used so the DNA was not entirely the same.
This is joining bits of DNA together (sometimes from different species). These are then inserted into a organism to produce a useful protein.
Recombinant DNA technologies
These are circular pieces of double stranded DNA. They are the critical elements of recombinant DNA technologies.
Plasmids
True or false. Plasmids are only found in some eukaryotes and all prokaryotes.
False.
True or false. Plasmids provide antibiotic resistance to hosts.
True (which gives selective advantage for bacteria)
This is a component of the plasmid that allows initiation of replication using host, DNA polymerase.
Origin of replication (Ori)
This is a component of the plasmid that allows selection of cells containing plasmid.
Antibiotic resistance gene
This is a component of the plasmid that drives the expression of your favourite gene in cells with appropriate transcription factor machinery. (These have to be specific to the gene/organism)
Promoter (animal-specific)
Why are there different promoters of the same gene in different organisms?
Because it is expressed in different cell types (like, the same gene but in a fish cell, or the same gene but in a neuron)
- it is just because the gene is expressed in a different cell type.
What are the 3 key components of plasmids?
Origin of replication, promoter, and the antibiotic resistance gene
These are responsible for cutting dsDNA (double stranded) at specific sequences.
Restriction enzymes
True or false. Bacteria do not have restriction enzymes, only humans.
False. Restriction enzymes are naturally found in bacteria which makes them such good plasmids.
What other function do restriction enzymes have in bacteria?
They provide defense system to degrade foreign DNA.
This is responsible for catalysing the formation of phosphodiester bond to repair nick in DNA backbone. It has complementary base pairing functions.
DNA ligase
This is the process of transferring plasmids into bacteria. recap: what’s a plasmid?
Transformation
A plasmid is a circular double stranded piece of DNA
These can replicate independently of the host’s chromosomal DNA.
Plasmids.
Outline the process of amplifying plasmids.
1) Transformed bacteria selected by antibiotic resistance gene.
2) Expression of plasmid gene in bacteria (through bacterial promoter)
3) Amplification of bacteria and purification of DNA (origin of replication)
What does the universal genetic code mean?
All organisms read the same codons as the same amino acids.
What is the significance of the genetic code being universal?
It means we can transform a human gene into bacteria and make it produce the same protein.
How can it produce the same protein? Because bacterial machinery can read the same codons and express them as the same proteins (as is in humans)
What is the main problem with human genes/proteins being processed in prokaryotes/bacteria?
Prokaryotes don’t have introns- so do not have the machinery to splice them out of the coding sequence.
True or false. A universal genetic code enables only some genes from one species to be expressed in another species.
False.
