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 A Toyota! Race fast, safe car. A Toyota GAATTC CTTAAG 5’ 3’ 5’ | | | “Restriction site” or “Recognition Sequence”

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

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

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

HOW ARE RE’S NAMED? After bacteria which produces them. GenusSpeciesStrain Order Isolated Escherichia coli R I EcoRI Haemophilus influenzae d III HindIII Bacillus amylo. H I BamHI Recognition SiteG^AATTC A^AGCTTG^GATGC

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.

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

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

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

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? 4 6 = once every 4096 bp Lambda (48,514 bp) would expect about 12 EcoRI sites

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?

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