Restriction EnzymesRestriction Enzymes

DiscoveryDiscovery

In 1962, Werner Arber, a Swiss In 1962, Werner Arber, a Swiss biochemist, provided the first evidence for biochemist, provided the first evidence for the existence of "molecular scissors" that the existence of "molecular scissors" that could cut DNA.could cut DNA.

He showed that He showed that E. coliE. coli bacteria have an bacteria have an enzymatic “immune system” that enzymatic “immune system” that recognizes and destroys foreign DNA, and recognizes and destroys foreign DNA, and modifies native DNA to prevent self-modifies native DNA to prevent self-destruction.destruction.

Molecular ScissorsMolecular Scissors

By the early 1970s these enzymes started By the early 1970s these enzymes started to be identified and purified.to be identified and purified.

It was shown that each species of bacteria It was shown that each species of bacteria had its own population of a SPECIFIC had its own population of a SPECIFIC restriction enzyme.restriction enzyme.

Each enzyme recognized its own specific Each enzyme recognized its own specific sequence of DNA bases. It is at this sequence of DNA bases. It is at this sequence that the DNA was cut.sequence that the DNA was cut.

Restriction Enzyme Recognition Restriction Enzyme Recognition SequencesSequences

What do the following phrases have in What do the following phrases have in common?:common?: Dammit, I'm mad!Dammit, I'm mad! Doc, note I dissent: a fast never prevents a Doc, note I dissent: a fast never prevents a

fatness. I diet on cod. fatness. I diet on cod. Dog DNA and GodDog DNA and God

Restriction enzymes usually recognize Restriction enzymes usually recognize palindromes in the nucleotide sequencespalindromes in the nucleotide sequences

Restriction Enzyme EcoRIRestriction Enzyme EcoRI Eco RI recognizes the sequence 5’….GAATTC…..Eco RI recognizes the sequence 5’….GAATTC….. A cut is made between the G and the A on each strand. A cut is made between the G and the A on each strand. This restriction enzyme leaves the nucleotides 5’AATT This restriction enzyme leaves the nucleotides 5’AATT

overhanging.overhanging. These are known as “sticky ends” because hydrogen bonds are These are known as “sticky ends” because hydrogen bonds are

available to “stick” to a complimentary 3’TTAAavailable to “stick” to a complimentary 3’TTAA Note: Restriction enzymes don’t stop with one cut! They Note: Restriction enzymes don’t stop with one cut! They

continue to cut at every recognition sequence on a DNA strand.continue to cut at every recognition sequence on a DNA strand.

Restriction Enzyme Cut from EcoRI

Videos and AnimationsVideos 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

Restriction enzymes, DNA, and Restriction enzymes, DNA, and ElectrophoresisElectrophoresis

• DNA normally comes in “Genome sized” lengths (usually several million bp in length.)

• These are the “elephants” in the race through the agarose and cant enter the gel matrix when they are this big.

• Restriction enzymes made possible the cutting of DNA into smaller fragments together with their separation and visualization by agarose gel electrophoresis.

Restriction Sites as “Molecular Restriction Sites as “Molecular Signposts”Signposts”

• Using two, or more different restriction enzymes on a DNA fragment enables those restriction sites to be mapped onto that DNA fragment.

EcoEco

Eco DigestEco DigestEcoEco cuts to cuts to yield two yield two DNA DNA fragmentsfragments

EcoEco

EcoEco

BglBgl

BglBgl

OrOr

Bgl DigestBgl DigestBglBgl also cuts also cuts to yield two to yield two DNA DNA fragments. fragments. But where is But where is the the BglBgl site in site in relation to the relation to the EcoEco site? site?

EcoEco BglBgl

EcoEco Bgl Bgl DoubleDouble Digest Digest

Shows it must be: Shows it must be:

A restriction A restriction digest with digest with both both EcoEco and and BglBgl enzymes enzymes provides the provides the answer.answer.

Your Turn:Your Turn:

• DNA- Off to the Races• Restriction Enzyme mapping challenge.