Restriction Enzymes Biotechnology Fall 2013.

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Presentation transcript:

Restriction Enzymes Biotechnology Fall 2013

Restriction Enzymes Restriction enzymes are protein enzymes produced by bacterium also known as 5’ – 3’restriction endonucleases recognize a specific sequence of nucleotides in double-stranded DNA Why? What about self-mutilation?

History In 1978 the Nobel prize was awarded to WERNER ARBER , DANIEL NATHANS and HAMILTON O. SMITH for the discovery of restriction enzymes and their application to problems of molecular genetics. The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetics

Restriction Enzymes Each restriction enzyme is specific for a certain combination or sequence of nucleotides (their restriction site) Scan DNA looking for the restriction site (the specific arrangement of nucleotides) Once the recognition sequence is found, the DNA molecule is cut by the enzyme at that site in a predictable manner

Blunt vs. Staggered Ends Restriction enzymes can leave blunt or staggered (“sticky”) ends at cutting sites 5’ CCC GGG GGG CCC 3’ 5’ GAATTC CTTAAG 3’ CCC GGG GGG CCC G AATTC CTTAA G Blunt ends Staggered Ends

 Restriction Map Lambda bacteriophage Very Well Know Restriction Sites

 DNA Marker Map Measure the actual mobility (distance migrated) of the fragments seen in the picture of the gel. Measure from the bottom of the well to the mid point of the band

 Restriction Map Make a graph of size vs.mobility of the DNA fragments on the semilog paper. This is your standard curve. Note: Do Not Connect Dots as in this picture, but make a Best Fit Line

 Restriction Map Generate a  Hind III restriction map by labeling all bands in your picture of the gel This will serve as a reference for other digests

Lab Experiment We will be digesting Lambda DNA with three restriction enzymes: HindIII EcoR1 Pst1 The digest with HindIII will serve as the marker We will find out how many EcoR1 and Pst1 restriction sites are in Lambda DNA, and what size fragments are created

Lab Experiment Steps Digest Lambda with the restriction enzymes Make a standard curve of HindIII data Use the standard curve of HindIII to analyze the digest with EcoR1 and Pst1