Restriction EnzymesRestriction Enzymes

Remember what we know Remember what we know about DNA.about DNA.

What is the monomer of DNA?What is the monomer of DNA? How do bases pair? How do bases pair? What kind of bond is used?What kind of bond is used?

Restriction EnzymesRestriction Enzymes Aka Restriction Aka Restriction

EndonucleasesEndonucleases What macromolecule do What macromolecule do

you think they are you think they are made of?made of?– Right, they are PROTEINS Right, they are PROTEINS

that cut strands of DNA that cut strands of DNA at specific nucleotide at specific nucleotide sequencessequences

Restriction EnzymesRestriction Enzymes

There are many different restriction There are many different restriction enzymes that each cut DNA at enzymes that each cut DNA at different nucleotide sequencesdifferent nucleotide sequences

Most will cut the DNA with a Most will cut the DNA with a staggered cutstaggered cut

Usually occurs at a Usually occurs at a palindromepalindrome5'GAATTC 3'CTTAAG

Sticky endsSticky ends The staggered cuts leave the DNA The staggered cuts leave the DNA

with end pieces “sticking off” with end pieces “sticking off” – We call these “sticky ends”We call these “sticky ends”– These exposed N-bases will want to join These exposed N-bases will want to join

with other complimentary exposed with other complimentary exposed bases bases

What if???What if???

What do you predict could happen if What do you predict could happen if two pieces of DNA are cut with the two pieces of DNA are cut with the same restriction enzyme???same restriction enzyme???– YES! They will have the same “sticky YES! They will have the same “sticky

ends”ends”

– How could we use this???How could we use this???

Restriction Enzymes -KindsRestriction Enzymes -Kinds Sticky End- already discussedSticky End- already discussed Blunt End Blunt End

– These cut the DNA straight across and These cut the DNA straight across and create blunt ends:create blunt ends:

– CCCGGGCCCGGG

GGGCCCGGGCCC

Products generated by restriction enzymes

COHESIVE END CUTTERS (staggered cuts):Enzyme Recognition Site Ends of DNA After Cut

EcoRI 5’…GAATTC…3’ 5’…G AATTC…3’3’…CTTAAG…5’ 3’…CTTAA G…5’

PstI 5’…CTGCAG…3’ 5’…CTGCA G…3’3’…GACGTC…5’ 3’…G ACGTC…5’

BLUNT END CUTTERS (direct cuts):Enzyme Recognition Site Ends of DNA After Cut

HaeIII 5’…GGCC…3’ 5’…GG CC…3’3’…CCGG…5’ 3’…CC GG…5’

Restriction enzymes are named according to the following nomenclature:

Ex: EcoRI E = genus Escherichia

co = species coli R = strain RY13

I = first enzyme isolated

In case you were curious …

Why would anyone go through Why would anyone go through the trouble of cutting DNA???the trouble of cutting DNA???

One reason…One reason…– Recombinant DNARecombinant DNA

Break down the word…what do you think Break down the word…what do you think recombinant means?recombinant means?

– Other reasons…DNA fingerprinting, gene Other reasons…DNA fingerprinting, gene therapy…therapy…

DNA that has been cut from one DNA that has been cut from one strand of DNA and then inserted into strand of DNA and then inserted into the gap of another piece of DNA that the gap of another piece of DNA that has been broken.has been broken.– The host DNA is often a bacterial cell The host DNA is often a bacterial cell

such as such as E coliE coli. .

Bacteria are often used in biotechnology Bacteria are often used in biotechnology because they have plasmidsbecause they have plasmids

A plasmid is a circular A plasmid is a circular

piece of DNA that exists piece of DNA that exists

apart from the apart from the

chromosome and chromosome and

replicates independently of it.replicates independently of it.

The Plasmid is then called a The Plasmid is then called a VECTORVECTOR

What is a vector?What is a vector?– Something that is used to transfer genes Something that is used to transfer genes

into a host cellinto a host cell

Ex’sEx’s– BacterialBacterial

plasmidsplasmids– VirusesViruses

So how do I isolate a gene of So how do I isolate a gene of interest?interest?

Use a restriction enzyme!!! (duh!)Use a restriction enzyme!!! (duh!)

What next???What next???

Once the gene is isolated, how do we Once the gene is isolated, how do we join it with the organism’s DNA?join it with the organism’s DNA?

1. Cut the organism’s DNA with the 1. Cut the organism’s DNA with the same restriction enzyme…why?same restriction enzyme…why?– The sticky ends will naturally be The sticky ends will naturally be

attracted to each otherattracted to each other

2. Add DNA LIGASE: an enzyme that 2. Add DNA LIGASE: an enzyme that seals the fragments togetherseals the fragments together

What is this organism now What is this organism now called?called?

Transgenic OrganismTransgenic Organism- organisms - organisms that contain functional recombinant that contain functional recombinant DNA (rDNA) from a different DNA (rDNA) from a different organismorganism

What’s the point?What’s the point? Recombinant DNA has been gaining importance Recombinant DNA has been gaining importance

over the last few years, and will become more over the last few years, and will become more important as genetic diseases become more important as genetic diseases become more prevalent and agricultural area is reduced. Below prevalent and agricultural area is reduced. Below are some of the areas where Recombinant DNA are some of the areas where Recombinant DNA will have an impact:will have an impact:

– Better Crops (drought & heat resistance) Better Crops (drought & heat resistance) – Recombinant Vaccines (i.e. Hepatitis B) Recombinant Vaccines (i.e. Hepatitis B) – Production of clotting factors Production of clotting factors

– Production of insulin Production of insulin – Production of recombinant pharmaceuticals Production of recombinant pharmaceuticals – Plants that produce their own insecticides Plants that produce their own insecticides – Germ line and somatic gene therapy Germ line and somatic gene therapy

RECAPRECAP Steps for making a Steps for making a

transgenic organism:transgenic organism:1.1. Locate and isolate the Locate and isolate the

gene of interestgene of interest2.2. Cut out the gene and Cut out the gene and

cut the plasmid using cut the plasmid using the appropriate the appropriate restriction enzymerestriction enzyme

3. Insert the desired gene into the 3. Insert the desired gene into the plasmid matching up the sticky endsplasmid matching up the sticky ends

4. Use the enzyme DNA ligase 4. Use the enzyme DNA ligase to seal up the sticky endsto seal up the sticky ends

5. Transfer the vector in the host 5. Transfer the vector in the host organism where it will replicateorganism where it will replicate

6. Host organism produces the protein 6. Host organism produces the protein coded for by the recombinant DNAcoded for by the recombinant DNA

Insulin ProductionInsulin Production