WHAT ARE RESTRICTION WHAT ARE RESTRICTION ENZYMES?ENZYMES?

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

nuclenucleasease

Enzyme digests nucleic acids

WHAT DNA SEQUENCES DO WHAT DNA SEQUENCES DO RESTRICTION ENZYMES CUT?RESTRICTION ENZYMES CUT?Usually cut DNA at a “palindrome” such as Usually cut DNA at a “palindrome” such as GAATTC.GAATTC.

GAATTCCTTAAG

5’ 3’

3’ 5’

| | | | | | “Restriction site”or

“Recognition Sequence”

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

WHAT PARTS OF DNA ARE CUT?WHAT PARTS OF DNA ARE CUT?

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

WHAT’S LEFT AFTER RESTRICTION WHAT’S LEFT AFTER RESTRICTION ENZYMES CUT DNA?ENZYMES CUT DNA?

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

- Blunt cuts.

- Sticky ends

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

amyloliquefaciens

H

I

BamHI

Recognition Site G^AATTC A^AGCTT G^GATCC

WHAT MUST BE AVAILABLE FOR WHAT MUST BE AVAILABLE FOR RESTRICTION ENZYMES TO WORK?RESTRICTION ENZYMES TO WORK?

AllAll enzymesenzymes need the correct:need the correct:

Why are enzymes so needy?Why are enzymes so needy? Need the Need the same environment as the cell that same environment as the cell that produced them.produced them.

1.1. temperature temperature

2. pH2. pH

3. salt concentration3. salt concentration ““buffer”buffer”

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

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

Examples:Examples:Bacteria with human insulin gene

Golden Rice with Vitamin A gene

Cows with human milk protein genesCows with human milk protein genes

Goats with spider silk genesGoats with spider silk genes

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

Forensics (not always Forensics (not always crime scenes)crime scenes)

Examples of Transgenic OrganismsExamples of Transgenic Organisms

““Transgenic” – moving genes across speciesTransgenic” – moving genes across species

Examples:Examples:Bacteria with human insulin geneBacteria with human insulin gene

Golden Rice with Vitamin A geneGolden Rice with Vitamin A gene

Cows with human milk protein genesCows with human milk protein genes

Goats with spider silk genesGoats with spider silk genes

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.