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Transcript
Restriction Enzymes AP Biology Unit 2 Images obtained without permission from http://w3.dwm.ks.edu.tw/bio/activelearner/14/images/ch14summary.gif and http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif and http://www.symmation.com/gallery/images/restriction-enzyme-ecorV-th.jpg Restriction Enzymes: Molecular Scissors • Restriction enzymes (endonuleases) cut DNA at specific sequences • What kinds of bonds are broken when restriction enzymes cut? – Covalent bonds (within a single strand) – Hydrogen bonds (between Hydrogen strands) as a result of the bond Covalent bond strands coming apart Image taken without permission from http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif Origins of Restriction Enzymes • Naturally found in different types of bacteria • Bacteria use restriction enzymes to protect themselves from foreign DNA • Bacteria have mechanisms to protect themselves from the actions of their own restriction enzymes • Have been isolated and sold for use in lab work Examples of Restriction Enzymes Enzyme Organism source Recognized Sequence EcoRI Escherichia coli 5' GAATTC 3’ 3' CTTAAG 5’ TaqI Thermus aquaticus 5' TCGA 3’ 3' AGCT 5’ HindIII Haemophilus influenzae BamHI Bacillus amyloliquefaciens 5'AAGCTT 3’ 3'TTCGAA 5’ 5' GGATCC 3’ 3' CCTAGG 5’ AluI Arthrobacter luteus 5' AGCT 3’ 3' TCGA 5’ Sticky Ends vs. Blunt Ends • When restriction enzymes cut, they produce either – Sticky ends (single stranded sections at the ends) – Blunt ends 5’ - - - G A A T T C - - - 3’ III I 3’ - - - C T T A A I I I G - - - 5’ Sticky ends Recombinant DNA • Recombinant DNA is constructed using restriction enzymes • Important: In order to join 2 pieces of DNA together they have to be cut by the same restriction enzyme – Why? – Otherwise, the sticky ends won’t match– DNA can’t bind together Recombinant Plasmids Step 1: Gene of interest and plasmid are cut with the same restriction enzyme Step 2: mix together Step 3: Add DNA Ligase to seal DNA back together Question… • What kinds of bonds does DNA Ligase form? – Covalent bonds (between nucleotides in a single strand of DNA) Putting It All Together Create recombinant plasmid Transform plasmid into E. coli Restriction Analysis • Using restriction enzymes to find out information about a piece of DNA • We can use restriction enzymes to find out – The size of a plasmid – If there are any restriction sites for a particular enzyme on a piece of DNA (ex. EcoRI) – How many restriction sites for a particular enzyme – Where the restriction sites are located