9.1 Manipulating DNA

KEY CONCEPT Biotechnology relies on cutting DNA at specific places.

WHAT ARE RESTRICTION WHAT ARE RESTRICTION ENZYMES?ENZYMES?

Restriction Enyzmes – molecular scissors Restriction Enyzmes – 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.

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.

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

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

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.

9.1 Manipulating DNA

Scientists use several techniques to manipulate DNA.

• Chemicals, computers, and bacteria are used to work with DNA.

• Scientists use these tools in genetics research and biotechnology.

9.1 Manipulating DNA

Restriction enzymes cut DNA.

• Restriction enzymes act as “molecular scissors.” – come from various types of bacteria– allow scientists to more easily study and manipulate

genes– cut DNA at a specific nucleotide sequence called a

restriction site

9.1 Manipulating DNA

• Different restriction enzymes cut DNA in different ways.

– each enzyme has a different restriction site

9.1 Manipulating DNA

– some cut straight across and leave “blunt ends”

– some make staggered cuts and leave “sticky ends”

9.1 Manipulating DNA

Restriction maps show the lengths of DNA fragments.

• Gel electrophoresis is used to separate DNA fragments by size.– A DNA sample is cut with restriction enzymes.– Electrical current pulls DNA fragments through a gel.

9.1 Manipulating DNA

– Smaller fragments move faster and travel farther than larger fragments.

– Fragments of different sizes appear as bands on the gel.

9.1 Manipulating DNA

• A restriction map shows the lengths of DNA fragments between restriction sites.

– only indicate size, not DNA sequence

– useful in genetic engineering

– used to study mutations