2 In vivo Gene cloning using a host NB! NOT the same as whole animal cloning!Gene of interest inserted into a vectorusually a plasmid (circular bacterial DNA molecules)using restriction enzymes and ligationPlasmid placed in a host cell (bacterium or yeast) which reproduces rapidly, cloning the desired geneNot all the bacteria cells take up the recombinant DNA, so the host cells are usually recognised by markers
3 Reverse Transcription Bacteria cannot remove intronsReverse transcription makes DNA from mRNA (which has already had the introns removed)Uses reverse transcriptase isolated from retrovirusesMakes cDNA (complementary) from the mRNA using a primerRetroviruses are unusual – they do not only use host enzymes
4 Bacteria Plasmids as vectors Can be taken up by any bacterial speciesContain genes for antibiotic resistance – resistance can be used to mark the host cells
5 Step 1: Cut the DNATarget DNA and plasmids mixed with restriction enzymesRestriction enzymeDNAPlasmid same cuts compatible sticky ends
6 Step 2: LigationCompatible sticky ends anneal using ligaseLIGASE
9 Limitations Some plasmids re-anneal with themselves Not all cells take up the recombined plasmidsGene 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.