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WHAT ARE RESTRICTION ENZYMES?

WHAT ARE RESTRICTION ENZYMES?. Restriction Enyzmes – molecular scissors able to cut DNA. 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

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WHAT ARE RESTRICTION ENZYMES?

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  1. WHAT ARE RESTRICTION ENZYMES? • Restriction Enyzmes – molecular scissors able to cut DNA

  2. 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’

  3. HOW DO RESTRICTION ENZYMES WORK? • RE’s cut DNA’s phosphodiester bonds and hydrogen bonds.

  4. HOW DO RESTRICTION ENZYMES WORK? • - RE’s generate two different types of “cuts” - Sticky ends - Blunt cuts.

  5. WHERE DO RE’S COME FROM? • Bacteria • “Immune system” to protect against bacteriophages (bacteria-infecting viruses like Lambda).

  6. 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

  7. 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.

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

  9. 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

  10. HOW IS DNA PASTED TOGETHER? • Ligase – another enzyme which reconnects phosphodiester bonds. RE Video restriction enzymes.exe Bill Nye on Restriction Enzymes

  11. 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

  12. 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?

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

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