Lecture 19 - recombinant DNA technology Flashcards
what is recombinant DNA technology
joining bits of DNA together (sometimes from different species), these are then inserted into an organism to produce (express) a useful protein
what are plasmids and where are they commonly found
- common in bacteria but also found in eukaryotes
- usually circular pieces of double stranded DNA
what do plasmids do
- replicate independently of host chromosomal DNA
- provide benefit to host
what are the key components of recombinant DNA plasmids
- antibiotic resistance
- promotor
- restriction sites
- gene
- Ori (origin of replication)
what is the Ori on a plasmid and what does it do
origin of replication = allows initiation of replication using hot DNA polymerase
what does the antibiotic resistance gene do
allows selections of cells containing plasmid
what does the promotor do
drives expression of your favourite gene (e.g insulin or GFP) in cells with the appropriate transcription factor machinery
how is the same gene expressed in different cell types
the promotor must change to allow expression in different cell types e.g prokaryotic, eukaryotic, or cell type specific
what are used to ‘cut and paste’ DNA
restriction enzymes and DNA ligase
what do restrcition enzymes do
cuts dsDNA at specific sequences, each enzyme will cut a different piece of DNA
where are restriction enzymes naturally found
bacteria
what does DNA ligase do
allows complementary ends to bind (complementary base pairing)
what is transformation
transfer of plasmids into bacteria
what occurs when plasmid has been transformed into bacterial cell
the plasmid in cell expresses antibiotic resistance gene and when plated on agar antibiotic, only cells that contain the plasmid will be able to grow and divide
what is a stop codon
UGA
