1. Gene transfer
© 2016 Paul Billiet ODWS

2. Molecular scissors Restriction endonucleases
Enzymes used by bacteria to detect and cut out viral genes
They restrict the number of different types of viruses that can infect a species of bacterium
Several hundred isolated
Each type identifies a specific sequence of DNA
Usually produce a staggered cut leaving a “sticky end”.
© 2016 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’
© 2016 Paul Billiet ODWS

4. EcoRI cleaving DNA
© 2016 Paul Billiet ODWS

5. 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.
© 2016 Paul Billiet ODWS

6. Plasmids Small pieces of circular DNA Found in bacteria
Easily transferred from bacterium to bacterium
Not necessarily from the same species
Useful vector for transfer of genes
Insert desired gene into plasmid
Insert plasmid into host cell.
Bacterial plasmid
© 2016 Paul Billiet ODWS

7. Molecular glue DNA ligase sticks ends of DNA together
© 2016 Paul Billiet ODWS

8. 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
© 2016 Paul Billiet ODWS

9. 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).
© 2016 Paul Billiet ODWS

10. The next revolution in gene technology
Cutting DNA using restriction enzymes is easy but limited
The library of different enzymes is large but finite (>3000)
Wouldn’t it be nice to cut where you want…
© 2016 Paul Billiet ODWS

11. Sticky fingers
Zinc Finger Nucleases (ZFNs) can bind specifically with a sequence of DNA and cut it
This has been used to precipitate mutations.
© 2016 Paul Billiet ODWS
Zinc fingers (blue) stuck to DNA and RNA

12. TALENs
Transcription Activator-like Effectors (TALEs) are used by bacteria to help them infect plant cells
TALEs can be synthesised to bind to any given sequence of DNA
Combined with an endonuclease (TALEN) they can cut the DNA.
© 2016 Paul Billiet ODWS
A TALEN wrapped around a DNA molecule (orange)

13. Both ZFNs and TALENs need a custom protein to be synthesised
This is a lengthy process
It is much easier to synthesise a stretch of DNA or RNA.
© 2016 Paul Billiet ODWS

14. a CRISPR solution
Clustered regularly-interspaced short palindromic repeats (CRISPR)
Segments of DNA found in Prokaryotes
Consist of repeats and a spacer
The spacer comes from infections of the bacterium by viruses or plasmids
Genes for nucleases, called Cas proteins, are associated with CRISPR
Guided by an RNA transcript of the CRISPR sequence, the nuclease cuts out the viral segment
CRISPR/Cas is an immune system used by prokaryotes.
© 2016 Paul Billiet ODWS

15. a CRISPR solution
CRISPR locates a particular nucleotide sequence and Cas cuts it
Designer CRISPR/Cas9 systems can be made very easily…
…much more easily than ZFNs and TALENs.
A Cas nuclease (blue) with a CRISPR RNA (red) identifying and processing a piece of DNA (yellow)
© 2016 Paul Billiet ODWS