Lecture 19 - Recombinant DNA technology Flashcards
DIABETES
Type 1
Type 2
NZ Type 1
1:5000
childhood onset
NZ Type 2
15% lifelong risk
What are recombinant DNA technologies?
Joining bits of DNA together (some from different species).
Insert into organism to produce (express) a useful protein.
recombinant DNA technologies examples
Fluorescent jellyfish
Take DNA (green fluorescent protein) from jellyfish and place inside fly
what are critical elements for recombinant DNA technologies?
Plasmids
Plasmids
• circular pieces of double stranded DNA
• Replicate independently of
the host’s chromosomal
DNA
• Provide a benefit to hosts
e.g. antibiotic resistance
• Common in bacteria, but
also found in eukaryotes.
Key components of recombinant DNA plasmids
Origin of replication (ORI)
Antibiotic resistance gene
Promoter
Origin of replication (ORI)
allows initiation of replication using host DNA polymerase
Antibiotic resistance gene
allows selection of cells containing plasmid.
Promoter
Drives expression of your favorite gene (e.g. insulin or GFP) in cells with appropriate transcription factor.
Promoter needs to
change to allow expression in.
Bacteria (prokaryote)
Fish (Eukaryote)
Neurons (cell type specific)
Cutting and pasting DNA into plasmids
Restriction enzymes
Ligases
Restriction enzymes
- found in bacteria – defense system to degrade foreign DNA
* Cut dsDNA at specific sequences
DNA Ligases
- Complementary base pairing
* Catalyses formation of phosphodiester bond to repair nick in DNA backbone
Amplifying plasmids
transformation
Transformation
transfer of plasmids into bacteria
Transformed bacteria selected by
antibiotic resistance contained on plasmid
Amplification of bacteria and purification of DNA for downstream uses e.g.
PCR,
cloning, transfection into other cells or organisms
Expression of plasmid gene in bacteria
if bacterial promoter
The universal genetic code
Do All organisms ”read” the same codons as the same amino acids?
Yes
AUG
methionine
UGA
stop codon
If we transform a human gene into bacteria will it still make the same protein?
Yes
The catch when cloning eukaryotic genes for expression in prokaryotes
Use coding sequence only!
Why do we Use coding sequence only when cloning eukaryotic genes for expression in prokaryotes?
Prokaryotic genes
• Don’t have introns
• don’t have machinery to process eukaryotic introns
Recombinant DNA technologies combine
DNA from different species.
Restriction enzymes and DNA ligase are used
to manipulate DNA and bacteria are used to amplify DNA
A universal genetic code allows
genes from one species to be expressed in another species –
