In vivo Gene cloning using a host
NB! NOT the same as whole animal cloning! Gene of interest inserted into a vector usually a plasmid (circular bacterial DNA molecules) using restriction enzymes and ligation Plasmid placed in a host cell (bacterium or yeast) which reproduces rapidly, cloning the desired gene Not all the bacteria cells take up the recombinant DNA, so the host cells are usually recognised by markers
Bacteria cannot remove introns Reverse transcription makes DNA from mRNA (which has already had the introns removed) Uses reverse transcriptase isolated from retroviruses Makes cDNA (complementary) from the mRNA using a primer Retroviruses are unusual – they do not only use host enzymes
Bacteria Plasmids as vectors
Can be taken up by any bacterial species Contain genes for antibiotic resistance – resistance can be used to mark the host cells
Step 1: Cut the DNA Target DNA and plasmids mixed with restriction enzymes Restriction enzyme DNA Plasmid same cuts compatible sticky ends
Step 2: Ligation Compatible sticky ends anneal using ligase LIGASE
Step 3: Inserting plasmid into host cell
Step 3: Cloning to magnify
Limitations Some plasmids re-anneal with themselves
Not all cells take up the recombined plasmids Gene containing resistance to an antibiotic is often included with the inserted DNA – bacteria resistant to the anti-biotic have the recombinant DNA and will survive on a growing medium with antibiotic.
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