Unit 7- Using DNA Flashcards
What is a vector?
A length of DNA that carries the gene we want in a host cell
Why can’t we just insert a strand of DNA into a cell?
As its not incorporated into the cells genome the DNA will just get broken down
Shape and size of a plasmid
About 5 genes long and circular
What functions do vectors have?
- small but big enough to hold another gene
- closed loop so less likely to be broken down
- contains promoters so the gene gets replicated and expressed
- it contains marker genes so cells containing the vector can be seen
How is a gene inserted into a plasmid?
1- cut the desired gene from donor DNA using restriction endonucleases (sticky ends are needed)
2- use the same RE to cut the plasmid in the middle of a marker gene
3- mix the gene and plasmids in a test tube, as they were cut by the same RE they should have the same sticky ends and anneal by complementary base pairing
4- DNA ligase reforms the phosphodiester bonds to complete the hybrid vector
How can we insert modified DNA into bacteria?
Heat shock
Electroporation
How can we insert modified DNA into plant cells?
Gene gun
Plant infection
How can we insert modified DNA into humans and animals?
- micro injection
- liposomes
- viruses
What is heat shock?
Making the cell membrane permeable enough to insert the modified DNA by suddenly heating the cell
What is electroporation?
Temporarily disrupting the cell membrane using a high voltage pulse, so the membrane is permeable enough to let the plasmid in
How is DNA inserted via gene gun?
Firing a tiny gold particles coated in the modified DNA, using an air gun, plant cell walls are tough so this penetrates them
How is modified DNA inserted into a plant cell via plant infection?
Inserting a new gene into the plasmid of the bacteria that infects most species
Then exposing the pathogen to the plant
How is modified DNA inserted into animal cells via micro injection?
DNA is inserted directly into nucleus using a fine micro pipette
How is modified DNA inserted into an animal cell via liposomes?
Cells in vivo can fuse with liposomes which encase the DNA, hence inserting the DNA by endocytosis
How is modified DNA inserted into a human cell via a virus?
Viruses can be genetically modified to deliver the DNA instead of harmful virus DNA
What are marker genes?
Genes used to find which cells have been taken up the hybrid vector
What is the job of the first marker gene? And what is it normally
To distinguish which cells have and haven’t taken up the plasmid
And it’s a gene that has been artificially inserted into the plasmid eg antibiotic resistance
Give an example of what the first marker gene could be and how we could separate the cells which have taken it up and haven’t?
- the gene for tetracycline resistance
- grow the cells on a tetracycline agar, the ones which grow have taken in the plasmid
What is the job of the second marker gene?
To distinguish between which cells have taken in a hybrid vector and unaltered vector (one which was broken and then annealed straight away)
Give an example of a second marker gene and how we can distinguish between cell which have hybrid vectors or normal plasmids?
- the second marker gene can be a gene for ampicillin resistance or a gene which expresses a flourescent protein
- the forgein gene is inserted in the middle of the second marker gene so that it no longer works, so the bacteria with the hybrid vector don’t have fluorescent proteins or resistance to ampicillin
- we can work this out through replica plating
Why is replica plating important?
So we don’t kill all the bacteria with the hybrid vector
How does replica plating work?
- a master plate with the bacteria which have taken up the plasmid or hybrid vector is placed on a sterile velvet block
- this leaves an imprint of all the colonies
- imprint agar plates containing the second antibiotic and some without on the velvet
- the bacteria that grow on both have taken up an unmodified plasmid
- the bacteria that die on the antibiotic agar but not the normal one have the modified plasmid
What are some advantages and disadvantages of cloning genes through PCR?
- quick and automated
- high error rate due to no natural correcting mechanism
- can use DNA from most sources
What are some advantages and disadvantages of cloning genes using living cells
- complex and multi step, requiring many days to complete
- large amounts of original DNA needed as success rate is below 1%
- needs pure DNA, can’t just be taken from a crime scene
- low error rate due to availability of natural correcting system
