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Biotechniques. Restriction Enzymes. Bacteria have endonucleases to cut up viral DNA. About 400 different endonucleases Each endonuclease recognises a specific sequence of about 4-6 nucleotide bases Cut DNA into fragments at these recognition sites. Specific. - PowerPoint PPT Presentation
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Restriction Enzymes
Bacteria have endonucleases to cut up viral DNA.
About 400 different endonucleases
Each endonuclease recognises a specific sequence of about 4-6 nucleotide bases
Cut DNA into fragments at these recognition sites
Specific
• different enzymes cut the DNA at different places
• same enzymes always cut the DNA at the same places
• enzymes are named after the bacteria they are taken from
Protection against cutting
• Bacteria can protect their own DNA from restriction enzymes
• add methyl groups to A and C bases in sequences recognised by their own endonucleases
Genetic Engineer Toolkits
Recognition
Sitescuts
• isolate specific DNA fragments
• manipulate individual genes from any organism.
C T T A A
A A T T C G
G
Sticky Ends
FragmentRestriction
enzyme: EcoRI
C T T A A
A A T T C
G
G A A T T C
C T T A AG
G
C T T A A
A A T T C G
G
• Cut DNA with an overhang• Only complementary sticky ends
can join (same restriction enzyme)
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
Blunt Ends
can join to any other blunt end fragment - non-specific
cutcut
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
G G G
G G G
C C C
C C C
DNA
Restriction enzymes• http://videos.howstuffworks.com/hsw/22635-discoveries-with-bill-nye-re
striction-enzymes-video.htm (restriction enzymes, 1 min)
• http://www.dnatube.com/video/1008/Tissue-Culture (restriction enzymes, 1 min)
• http://www.youtube.com/watch?v=aA5fyWJh5S0 (restriction enzymes, 1 min – no sound)
• http://www.youtube.com/watch?v=AEINuCL-5wc&feature=related
(restriction enzyme, 3 min – very simple)
• http://www.youtube.com/watch?v=zF3REf7yKpw&feature=related (restriction enzymes, 7 min – students in lab, no sound??)