Transcript Powerpoint Presentation: Gene Transfer

Gene transfer
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”
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© 2010 Paul Billiet ODWS
Restriction enzymes
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Eco RI from Eschericia coli
5’ GAATTC 3’
3’ CTTAAG 5’
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Hind III from Hemophilous influenzae
5’ AAGCTT 3’
3’ TTCGAA 5’
© 2010 Paul Billiet ODWS
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
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© 2010 Paul Billiet ODWS
Plasmids
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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
© 2010 Paul Billiet ODWS
Image Credit: Bacterial plasmid
Splicing in the gene
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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
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)
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© 2010 Paul Billiet ODWS