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2017-04-04 Restriction Endonucleases! Mr.Masri SBI4U Humberside C. I. Restriction Endonucleases (RE)! ! also called restriction enzymes! ! naturally found in bacteria! ! used as a defense mechanism against bacteriophages by degrading their DNA! 1 2017-04-04 Restriction Endonucleases (RE)! ! The DNA of a bacterial cell is protected from the cell’s own RE by the addition of methyl groups to adenines or cytosines within the sequences recognized by the enzymes.! Restriction Enzymes (RE)! ! molecular scissors which bind to DNA at a specific base sequence (recognition site) and then cuts (or hydrolyzes) the phosphodiester bonds on both strands of DNA 2 2017-04-04 Restriction Enzymes (RE)! ! once the cuts have been made, the weakness of the hydrogen bonds holding the DNA fragments together allow the DNA fragments to separate Restriction Enzymes (RE)! ! Cuts are defined and reproducible (very important)! ! Each RE has its own unique recognition site (4-8 nucleotides long)! 3 2017-04-04 Palindromes! ! All recognition sites are palindromes, (read the same way forward and backward) ! Palindromes in English! ! Never odd or even! ! Tips spill, lips spit! ! Devil never even lived! ! Go hang a salami, I’m a lasagna hog! ! So many dynamos! ! Sit on a potato pan, Otis! ! Mr. Owl ate my metal worm! 4 2017-04-04 Palindromes in Molecular Biology! ! a DNA sequence can be read the same in the 5I à 3I direction on COMPLEMENTARY strands: 5 GCAATTGC 3 3 CGTTAACG 5 | | | | The Cutting…! When restriction enzymes cut phosphodiester bonds directly across each strand, blunt ends are generated.! GATC CTAG GA CT +! T C ! AG 5 2017-04-04 The Cutting…! When restriction enzymes cut phosphodiester bonds in a staggered (or offset) pattern across from each strand, sticky ends are generated.! G A A T T C! C T T A A G! ! A A T T C! G! +!! ! G! C T T A! A! ! ! Sticky Ends! ! Sticky ends are very useful because if two different pieces of DNA are cut with the same restriction enzyme, the overhanging sticky ends will complementarily base pair, creating a recombinant DNA molecule.! ! DNA ligase joins the fragments by producing the phosphodiester bonds between nucleotides! 6 2017-04-04 Significance of Sticky Ends Two different fragments cut by the same restriction enzyme have identical sticky ends and are able to join together How are RE named?! ! RE are named after the bacteria from which they were isolated.! ! Example: EcoRI E! !Escherichia (genus)! co !coli (species)! R! !RY13 (strain)! I ! !first endonuclease isolated! 7 2017-04-04 Examples! Applications of RE! (1) Forensics! • Inherited variations in base sequence lead to differences in places where DNA is cut • RFLPs (Restriction Fragment Length Polymorphisms) DNA from different individuals give different fragment lengths 8 2017-04-04 Applications of RE! Differences detected by cutting the DNA with specific RE’s and running the DNA fragments through gel electrophoresis Significance of RE! (2) Recombinant DNA Technology! • DNA from different sources are cut with the same RE and are then joined together! • Commonly used in used in amplifying DNA, GM foods, medicine and creating new DNA strands.! • E.g. human insulin inserted into a bacteria’s plasmid DNA using RE! !! 9 2017-04-04 Homework! ! Read pg. 266-267! ! Pg. 267 #1-5! ! Read pg. 278-284 ! ! pg. 281 #1, 2 and 5! ! pg. 282 #6, 9! 10