WHAT ARE RESTRICTION WHAT ARE RESTRICTION ENZYMES?ENZYMES?

Restriction Enyzmes Restriction Enyzmes – molecular scissors – molecular scissors able to cut DNAable to cut DNA

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

Usually cut DNA at a “palindrome” such as GAATTC.Usually cut DNA at a “palindrome” such as GAATTC.

Palindrome – word or phrase when spelled backwords, Palindrome – word or phrase when spelled backwords, spells the same word or phrasespells the same word or phrase

Ex. Ex. BOBBOBMADAM I’M ADAMMADAM I’M ADAMA Toyota! Race fast, safe car. A Toyota A Toyota! Race fast, safe car. A Toyota

GAATTCCTTAAG

5’ 3’

3’ 5’

| | | | | | “Restriction site”or

“Recognition Sequence”

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

RE’s cut DNA’s RE’s cut DNA’s phosphodiester bonds phosphodiester bonds and hydrogen bonds.and hydrogen bonds.

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

- RE’s generate two different types of “cuts”

- Sticky ends

- Blunt cuts.

WHERE DO RE’S COME FROM?WHERE DO RE’S COME FROM?

BacteriaBacteria

““Immune system” to Immune system” to protect against protect against bacteriophages bacteriophages (bacteria-infecting (bacteria-infecting viruses like Lambda).viruses like Lambda).

HOW ARE RE’S NAMED?HOW ARE RE’S NAMED?

After bacteria which produces them.After bacteria which produces them.

GenusGenus

SpeciesSpecies

StrainStrain

Order IsolatedOrder Isolated

Escherichia

coli

R

I

EcoRI

Haemophilus

influenzae

d

III

HindIII

Bacillus

amylo.

H

I

BamHI

Recognition Site G^AATTC A^AGCTT G^GATGC

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

Must provide correct temperature and Must provide correct temperature and buffer (salt, pH) for enzyme to work.buffer (salt, pH) for enzyme to work.

Mimics cellular conditions of bacteria they Mimics cellular conditions of bacteria they come from.come from.

WHAT ARE RE’S USED FOR?WHAT ARE RE’S USED FOR?

Genetic engineering – Genetic engineering – pasting together DNA pasting together DNA from two different from two different organisms. organisms.

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

Which are more useful in genetic Which are more useful in genetic engineering? RE’s that generate sticky ends engineering? RE’s that generate sticky ends or ones that produce blunt cuts?or ones that produce blunt cuts?

STICKY ENDS

HOW IS DNA PASTED HOW IS DNA PASTED TOGETHER?TOGETHER?

Ligase – another enzyme which Ligase – another enzyme which reconnects phosphodiester bonds.reconnects phosphodiester bonds.

RE Video

restriction enzymes.exe

Bill Nye on Restriction Enzymes

WHAT ELSE ARE RE’S USED WHAT ELSE ARE RE’S USED FOR?FOR?

Forensics – DNA Fingerprinting for crime Forensics – DNA Fingerprinting for crime scene investigation and paternity testing.scene investigation and paternity testing.

Everyone’s DNA has a different sequence Everyone’s DNA has a different sequence – even though only 0.1% different.– even though only 0.1% different.

How frequently would EcoRI cut DNA?How frequently would EcoRI cut DNA?4

6= once every 4096 bp

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

THOUGHT QUESTIONTHOUGHT QUESTION

Bacteria are prokaryotes.Bacteria are prokaryotes.

Prokaryotes do not have a nucleus.Prokaryotes do not have a nucleus.

Both DNA and RE’s are in cytoplasm.Both DNA and RE’s are in cytoplasm.

Why isn’t bacterial DNA cut by RE’s?Why isn’t bacterial DNA cut by RE’s?

MethylationMethylation

See boardSee board

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