1. Restriction Enzymes Aims: Must be able to recall the basic functions of restriction enzymes. Should be able to outline how specific restriction enzymes work. Could be able to explain the different ends produced by restriction enzymes and their benefits.

2. Restriction Enzymes RESTRICTION ENZYMES = one of the essential tools of genetic engineering. Purified forms of these naturally occurring bacterial enzymes are used as “molecular scalpels”. Allow genetic engineers to cut up DNA in a controlled way. Restriction enzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called RECOGNITION SITES. By using a ‘tool kit’ of over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers are able to isolate and sequence DNA

3. Restriction Enzymes Recognition Site GAATTC CTTAAG DNA CTTAAG GAATTC cut restriction enzyme EcoRI cuts here

4. Specific Recognition Sequences Named according to the bacterial species first isolated from. Followed by a number to distinguish different enzymes isolated from the same organism. e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H. A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site: EnzymeSource Recognition Sites EcoRI Escherichia coli RY13 GAATTC BamHI Bacillus amyloliquefaciens H GGATCC HaeIII Haemophilus aegyptius GGCC HindIII Haemophilus influenzae Rd AAGCTT Hpal Haemophilus parainfluenzae GTTAAC

5. C T T A A A A T T CG G Fragment Restriction enzyme: EcoRI Sticky endRestriction enzyme: EcoRI DNA from another source A restriction enzyme cuts the double- stranded DNA molecule at its specific recognition site The two different fragments cut by the same restriction enzyme have identical sticky ends and are able to join together The cuts produce a DNA fragment with two “sticky” ends When two fragments of DNA cut by the same restriction enzyme come together, they can join by base-pairing C T T A A A A T T C G G C T T A A G G A A T T CG G It is possible to use restriction enzymes that cut leaving an overhang; a so- called “sticky end”. DNA cut in such a way produces ends which may only be joined to other sticky ends with a complementary base sequence. Sticky Ends

6. C C C G G G C C C G G G C C C G G G C C C Blunt Ends   Possible to use restriction enzymes that cut leaving no overhang; a so-called “blunt end”.   DNA cut in such a way is able to be joined to any other blunt end fragment, but tends to be non- specific because there are no sticky ends as recognition sites. Restriction enzyme cuts here Recognition Site DNA from another source The cut by this type of restriction enzyme leaves no overhang cut C C C G G G C C C G G G C C C G G G C C C DNA A special group of enzymes can join the pieces together

7. Activity Read the information on p221 in Biozone. Answer questions on p222 in Biozone.