1. Biotechniques

2. 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

3. Reverse Transcription
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

4. 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

5. Step 1: Cut the DNA
Target DNA and plasmids mixed with restriction enzymes
Restriction enzyme
DNA
Plasmid
 same cuts  compatible sticky ends

6. Step 2: Ligation
Compatible sticky ends anneal using ligase
LIGASE

7. Step 3: Inserting plasmid into host cell

8. Step 3: Cloning to magnify

9. 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.