Restriction Enzymes

Discovery  In 1962, Werner Arber, a Swiss biochemist, provided the first evidence for the existence of "molecular scissors" that could cut DNA.  He showed that E. coli bacteria have an enzymatic “immune system” that recognizes and destroys foreign DNA, and modifies native DNA to prevent self- destruction.

Molecular Scissors  By the early 1970s these enzymes started to be identified and purified.  It was shown that each species of bacteria had its own population of a SPECIFIC restriction enzyme.  Each enzyme recognized its own specific sequence of DNA bases. It is at this sequence that the DNA was cut.

Restriction Enzyme Recognition Sequences  What do the following phrases have in common?: Dammit, I'm mad! Dammit, I'm mad! Doc, note I dissent: a fast never prevents a fatness. I diet on cod. Doc, note I dissent: a fast never prevents a fatness. I diet on cod. Dog DNA and God Dog DNA and God  Restriction enzymes usually recognize palindromes in the nucleotide sequences

Restriction Endonucleases Enzymes recognize specific 4-8 bp sequences Some enzymes cut in a staggered fashion - “sticky ends” EcoRI 5’…GAATTC…3’ EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’ 3’…CTTAAG…5’ Some enzymes cut in a direct fashion – “blunt ends” PvuII 5’…CAGCTG…3’ PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’ 3’…GTCGAC…5’

Restriction Endonucleases Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1

Restriction Enzyme EcoRI  Eco RI recognizes the sequence 5’….GAATTC…..  A cut is made between the G and the A on each strand.  This restriction enzyme leaves the nucleotides 5’AATT overhanging.  These are known as “sticky ends” because hydrogen bonds are available to “stick” to a complimentary 3’TTAA  Note: Restriction enzymes don’t stop with one cut! They continue to cut at every recognition sequence on a DNA strand. Restriction Enzyme Cut from EcoRI

Videos and Animations Click on “Techniques” then “Cutting and Pasting” and view the 2D animation and 3D Cartoon Video to see Restriction enzymes in action

Restriction enzymes, DNA, and Electrophoresis DNA normally comes in “Genome sized” lengths (usually several million bp in length.) These are the “elephants” in the race through the agarose and cant enter the gel matrix when they are this big. Restriction enzymes made possible the cutting of DNA into smaller fragments together with their separation and visualization by agarose gel electrophoresis.

Restriction Sites as “Molecular Signposts” Using two, or more different restriction enzymes on a DNA fragment enables those restriction sites to be mapped onto that DNA fragment.

Eco Eco Digest Eco cuts to yield two DNA fragments

Eco Eco Bgl Bgl Or Bgl Digest Bgl also cuts to yield two DNA fragments. But where is the Bgl site in relation to the Eco site?

Eco Bgl Eco Bgl Double Digest Shows it must be: A restriction digest with both Eco and Bgl enzymes provides the answer.

Your Turn: DNA- Off to the Races Restriction Enzyme mapping challenge.