3 KEY CONCEPT Biotechnology relies on cutting DNA at specific places.
4 WHAT ARE RESTRICTION ENZYMES? Restriction Enyzmes – molecular scissors able to cut DNA
5 HOW DO RESTRICTION ENZYMES WORK? Usually cut DNA at a “palindrome” such as GAATTC.Palindrome – word or phrase when spelled backwords, spells the same word or phraseEx. BOBMADAM I’M ADAMA Toyota! Race fast, safe car. A Toyota5’3’GAATTC| | | | | |“Restriction site”or“Recognition Sequence”CTTAAG3’5’
6 HOW DO RESTRICTION ENZYMES WORK? RE’s cut DNA’s phosphodiester bonds and hydrogen bonds.
7 HOW DO RESTRICTION ENZYMES WORK? - RE’s generate two different types of “cuts”- Sticky ends- Blunt cuts.
9 WHERE DO RE’S COME FROM? Bacteria “Immune system” to protect against bacteriophages (bacteria-infecting viruses like Lambda).
10 HOW ARE RE’S NAMED? After bacteria which produces them. Genus Species StrainOrder IsolatedEcoRIHindIIIBamHIEscherichiaHaemophilusBacilluscoliinfluenzaeamylo.RdHIIIIIRecognition SiteG^AATTCA^AGCTTG^GATGC
11 HOW DO RESTRICTION ENZYMES WORK? Must provide correct temperature and buffer (salt, pH) for enzyme to work.Mimics cellular conditions of bacteria they come from.
13 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.one hundredth of a percentage point
14 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.
15 Eco cuts to yield two DNA fragments Eco DigestEco
16 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
17 A restriction digest with both Eco and Bgl enzymes provides the answer. Eco Bgl Double DigestShows it must be:EcoBgl
18 Your Turn:DNA- Off to the RacesRestriction Enzyme mapping challenge.
19 WHAT ARE RE’S USED FOR?Genetic engineering – pasting together DNA from two different organisms.
20 HOW DO RESTRICTION ENZYMES WORK? Which are more useful in genetic engineering? RE’s that generate sticky ends or ones that produce blunt cuts?STICKY ENDS
21 HOW IS DNA PASTED TOGETHER? Ligase – another enzyme which reconnects phosphodiester bonds.RE Videorestriction enzymes.exeBill Nye on Restriction Enzymes
22 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
23 WHAT ELSE ARE RE’S USED FOR? Forensics – DNA Fingerprinting for crime scene investigation and paternity testing.Everyone’s DNA has a different sequence – even though only 0.1% different.How frequently would EcoRI cut DNA?46= once every 4096 bpLambda (48,514 bp) would expect about 12 EcoRI sites
24 THOUGHT QUESTION Bacteria are prokaryotes. Prokaryotes do not have a nucleus.Both DNA and RE’s are in cytoplasm.Why isn’t bacterial DNA cut by RE’s?
25 MethylationSee boardIn humans, methyl groups are used to tag genes to turn them on or off. Stay tuned.
26 Scientists use several techniques to manipulate DNA. Chemicals, computers, and bacteria are used to work with DNA.Scientists use these tools in genetics research and biotechnology.
27 Restriction enzymes cut DNA. Restriction enzymes act as “molecular scissors.”come from various types of bacteriaallow scientists to more easily study and manipulate genescut DNA at a specific nucleotide sequence called a restriction site
28 Different restriction enzymes cut DNA in different ways. each enzyme has a different restriction site
29 some cut straight across and leave “blunt ends” some make staggered cuts and leave “sticky ends”
30 Restriction maps show the lengths of DNA fragments. Gel electrophoresis is used to separate DNA fragments by size.A DNA sample is cut with restriction enzymes.Electrical current pulls DNA fragments through a gel.
31 Smaller fragments move faster and travel farther than larger fragments. Fragments of different sizes appear as bands on the gel.
32 A restriction map shows the lengths of DNA fragments between restriction sites. only indicate size, not DNA sequenceuseful in genetic engineeringused to study mutations