KEY CONCEPT Biotechnology relies on cutting DNA at specific places.

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 phrase Ex. BOB MADAM I’M ADAM A Toyota! Race fast, safe car. A Toyota 5’ 3’ GAATTC “Restriction site” or “Recognition Sequence” | | | | | | CTTAAG 3’ 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 • Strain • Order Isolated EcoRI HindIII BamHI Escherichia Haemophilus Bacillus coli influenzae amylo. R d H I III I Recognition Site G^AATTC A^AGCTT G^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.

12. If different alleles can be cut up and identified (using GE), we can identify unknown persons (e.g., criminals, biological relatives), and document unknown genotypes (e.g., Huntington’s Disease) http://www.youtube.com/watch?v=KVnFBCzg8Ms Why cut up DNA?

13. You have a vial of undescribed DNA fragments—now what? Samples pipetted into wells on one end of a gel (e.g., agarose) Electricity is added to the gel DNA fragments move through the gel at different rates, away from the negative and toward the positive end Smaller fragments move easier and further from well, larger fragments more cumbersome and move shorter distances from well The resulting “fingerprint” of fragments can be matched to a known sample or investigated for specific alleles Gel Electrophoresis

14. A B C D E F G

15. Eco Digest Eco cuts to yield two DNA fragments Eco

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

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

18. Restriction enzymes cut up DNA into pieces unique to your DNA DNA pieces are a function of your genetics and the restriction enzyme used DNA fragment soup placed in Gel well and distributes based on fragment/sequence length Resulting gel is unique—like a fingerprint Review

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

20. WHAT ARE RE’S USED FOR? • Genetic engineering – pasting together DNA from two different organisms.

21. 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

22. HOW IS DNA PASTED TOGETHER? • Ligase – another enzyme which reconnects phosphodiester bonds. RE Video

23. 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

24. 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 bp Lambda (48,514 bp) would expect about 12 EcoRI sites

25. 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?

26. Methylation • See board • In humans, methyl groups are used to tag genes to turn them on or off. Stay tuned.

27. 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.

28. Restriction enzymes cut DNA. • Restriction enzymes act as “molecular scissors.” • come from various types of bacteria • allow scientists to more easily study and manipulate genes • cut DNA at a specific nucleotide sequence called a restriction site

29. Different restriction enzymes cut DNA in different ways. • each enzyme has a different restriction site

30. some cut straight across and leave “blunt ends” • some make staggered cuts and leave “sticky ends”

31. 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.

32. Smaller fragments move faster and travel farther than larger fragments. • Fragments of different sizes appear as bands on the gel.

33. A restriction map shows the lengths of DNA fragments between restriction sites. • only indicate size, not DNA sequence • useful in genetic engineering • used to study mutations