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

• 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??)