2 DiscoveryIn 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.
3 Molecular ScissorsBy 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.
4 Restriction Enzyme Recognition Sequences What do the following phrases have in common?:Dammit, I'm mad!Doc, note I dissent: a fast never prevents a fatness. I diet on cod.Dog DNA and GodRestriction enzymes usually recognize palindromes in the nucleotide sequences
5 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’TTAANote: Restriction enzymes don’t stop with one cut! They continue to cut at every recognition sequence on a DNA strand.Model with velcro pieces to emphasize the sticky ends.Restriction Enzyme Cut from EcoRI
6 Videos and Animations http://www.dnai.org/b/ Click on “Techniques” then “Cutting and Pasting” and view the 2D animation and 3D Cartoon Video to see Restriction enzymes in action
7 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.
8 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.
9 Eco cuts to yield two DNA fragments Eco DigestEco
10 Bgl also cuts to yield two DNA fragments Bgl also cuts to yield two DNA fragments. But where is the Bgl site in relation to the Eco site?Bgl DigestBglEcoOrEcoBgl
11 A restriction digest with both Eco and Bgl enzymes provides the answer. Eco Bgl Double DigestShows it must be:EcoBgl
12 Your Turn:DNA- Off to the RacesRestriction Enzyme mapping challenge.