Lecture 19: RECOMBINANT DNA TECHNOLOGIES Flashcards
How many people get type 1 diabetes?
1 in 5000
When does type 1 diabetes develop?
Childhood usually
What is type 1 diabetes?
Loss of beta cells in the pancreas which make insulin
How many people get type 2 diabetes?
15%
When does type 2 diabetes develop?
Lifelong risk
What is type 2 diabetes?
Resistance to insulin
What happens in 1922?
Insulin first isolated from pig and cattle pancreas
What happened in the 1950’s?
Insulin proteins sequence/structure identified
What happened in 1973?
Recombinant DNA technology introduced (PCR and restriction enzymes)
What happened in 1977?
Insulin gene mapped to chromosome 11
What happened in 1980?
Gene for insulin sequenced
What happened in 1982?
Eli Lilly produced first recombinant insulin
Where was insulin purified from?
The pancreas of cattle or pigs
How does of insulin compare between humans, cows and pigs?
The amino acid sequences are similar but not identical
What is the structure of insulin?
Alpha and beta chains held together by disulfide bonds (1 between the two chains and one within the alpha chain)
How many differences between human and cow insulin?
2
How many differences between human and pig insulin?
1
What happens when cow or pig insulin is introduced into humans?
Immune responses range from local irritation to anaphylactic shock
What is the problem with cow and pig insulin?
It isn’t always 100% pure
What has also been used as therapeutic proteins?
Human sources but there are issues arounds safety (pathogen transmission), yields and source of a protein (tissue availability)
What are recombinant DNA technologies?
Joining bits of DNA together (sometimes from different species). These are inserted into an organism to produce (express) a useful protein
What is the structure of plasmids?
Usually circular pieces of double stranded DNA found independently of the chromosome
How do plasmids replicate?
Independently of the hosts chromosomal DNA but uses all the cells machinery
Where are plasmids found?
Common in bacteria, but also found in eukaryotes and some fungi
What do plasmids provide?
A benefit to hosts - antibiotic resistance
What are the key components of recombinant DNA using plasmids?
Origin of replication (ORI), antibiotic resistance gene and promoter
What does the ORI allow?
Initiation of replication using host DNA polymerase
What does the antibiotic resistance gene allow?
Selection of cells containing plasmid
What does the promoter do?
Drives expression of your favourite gene in cells with appropriate transcription factor machinery
What must the promoter do?
It needs to change to allow expression in prokaryotes, eukaryotes and specific cell types
What is used to cut DNA?
Restriction enzymes
Where are restriction enzymes found?
Naturally in bacteria, used as a defence system to degrade foreign DNA
What do restriction enzymes do?
Cut the double stranded DNA of the plasmid at specific sequences
How is the DNA insert made?
To match the cut of the DNA backbone with complementary base pairing
What does DNA Ligase do?
Catalyses the formation of phosphodiester bonds to repair nick in DNA backbone
What is transformation?
Transfer of plasmids into bacteria
What are transformed bacteria selected by?
Antibiotic resistance contained on plasmid
What happens after the transformed bacteria are selected?
Expression of plasmid gene in bacteria (if bacterial promoter)
What happens after expression of plasmid gene in bacteria?
Amplification of bacteria and purification of DNA for downstream uses - PCR, cloning, transfection into other cells or organisms
What do all organisms do?
Read the same codons as the same amino acid
What is AUG?
Methionine
What is UGA?
Stop codon
What is the significance of the universal genetic code?
We can transform a human gene into bacteria and it will still make the same protein
What don’t prokaryote genes have?
Introns
What don’t prokaryotes have?
The machinery to process eukaryotic introns (can’t do splicing)
What can be used when making recombinant proteins in bacteria?
Only the coding sequence
What is used when making recombinant proteins?
The mRNA from the gene is copied and inserted into the plasmid as it doesn’t have introns