1. Gene transfer

2. Molecular scissors Restriction endonucleases
Enzymes used by bacteria to detect and cut out viral genes
Several hundred isolated
Each type identifies a specific sequence of DNA
Usually produce a staggered cut leaving a “sticky end”
© 2010 Paul Billiet ODWS

3. Restriction enzymes Eco RI from Eschericia coli 5’ GAATTC 3’
3’ CTTAAG 5’
Hind III from Hemophilous influenzae
5’ AAGCTT 3’
3’ TTCGAA 5’
© 2010 Paul Billiet ODWS

4. Identifying a gene
mRNA from cells making the desired protein is extracted
Reverse transcriptase used to make cDNA
cDNA used to make gene probes
Gene located on a chromosome
Gene sequenced
Gene bracketed by sequences cut by a restriction enzyme
Gene cut out using restriction enzyme
© 2010 Paul Billiet ODWS

5. Plasmids Small pieces of circular DNA Found in bacteria
Easily transferred from bacterium to bacterium
Not necessary from the same species
Useful vector for transfer of genes
Insert desired gene into plasmid
Insert plasmid into host cell
Image Credit: Bacterial plasmid
© 2010 Paul Billiet ODWS

6. Splicing in the gene
Sticky ends permit the fragment to be “glued” into another piece of DNA cut by the same enzyme
Gene
Sticky end
Sticky end
EcoRI
Ligase
© 2010 Paul Billiet ODWS

7. Gene expression Plasmid introduced into bacterial cell
Every time the bacterium divides the plasmid is replicated too
Gene expressed by the bacterium
Same protein is synthesised
Universal genetic code
Human proteins can be produced by bacteria
E.g. Humulin (Human Insulin) E.g. Human somatotropin (growth hormone)
© 2010 Paul Billiet ODWS