Gene transfer 2016 Paul Billiet ODWS Molecular scissors

Gene transfer © 2016 Paul Billiet ODWS

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

Restriction enzymes p Eco RI from Eschericia coli 5’ GAATTC 3’ 3’ CTTAAG 5’ p Hind III from Hemophilous influenzae 5’ AAGCTT 3’ 3’ TTCGAA 5’ © 2016 Paul Billiet ODWS

Eco. RI cleaving DNA © 2016 Paul Billiet ODWS

Identifying a gene m. RNA from cells making the desired protein is extracted p Reverse transcriptase used to make c. DNA p c. DNA used to make gene probes p Gene located on a chromosome p Gene sequenced p Gene bracketed by sequences cut by a restriction enzyme p Gene cut out using restriction enzyme. p © 2016 Paul Billiet ODWS

Plasmids p p p p 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. © 2016 Paul Billiet ODWS Bacterial plasmid

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

Splicing in the gene p Sticky ends permit the fragment to be “glued” into another piece of DNA cut by the same enzyme. Gene Sticky end Eco. RI Ligase © 2016 Paul Billiet ODWS

Gene expression Plasmid introduced into bacterial cell p Every time the bacterium divides the plasmid is replicated too p Gene expressed by the bacterium p Same protein is synthesised p Universal genetic code p Human proteins can be produced by bacteria p E. g. Humulin (Human Insulin) E. g. Human somatotropin (growth hormone). p © 2016 Paul Billiet ODWS

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

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

TALENs p p p 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)

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

a CRISPR solution p p p p 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 © 2016 Paul Billiet ODWS prokaryotes.

a CRISPR solution p p p CRISPR locates a particular nucleotide sequence and Cas cuts it Designer CRISPR/Cas 9 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