9.1 Manipulating DNA

KEY CONCEPT Biotechnology relies on cutting DNA at specific places.

9.1 Manipulating DNA

WHAT ARE RESTRICTION ENZYMES?

Restriction Enyzmes – molecular scissors able to cut DNA

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HOW DO RESTRICTION ENZYMES WORK?

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

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

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

GAATTCCTTAAG

5’ 3’

3’ 5’

| | | | | | “Restriction site”or

“Recognition Sequence”

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HOW DO RESTRICTION ENZYMES WORK?

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

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HOW DO RESTRICTION ENZYMES WORK?

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

- Sticky ends

- Blunt cuts.

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WHERE DO RE’S COME FROM?

Bacteria

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

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HOW ARE RE’S NAMED?

After bacteria which produces them.

Genus

Species

Strain

Order Isolated

Escherichia

coli

R

I

EcoRI

Haemophilus

influenzae

d

III

HindIII

Bacillus

amylo.

H

I

BamHI

Recognition Site G^AATTC A^AGCTT G^GATGC

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HOW DO RESTRICTION ENZYMES WORK?

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

Mimics cellular conditions of bacteria they come from.

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Why cut up DNA?• If different alleles can be cut up and identified (using

GE), we can identify unknown persons (e.g., criminals, biological relatives), and document unknown genotypes (e.g., Huntington’s Disease)

• http://www.youtube.com/watch?v=KVnFBCzg8Ms

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Gel Electrophoresis• You have a vial of undescribed DNA fragments—now

what?• Samples pipetted into wells on one end of a gel (e.g.,

agarose)• Electricity is added to the gel• DNA fragments move through the gel at different rates,

away from the negative and toward the positive end– Smaller fragments move easier and further from well,

larger fragments more cumbersome and move shorter distances from well

• The resulting “fingerprint” of fragments can be matched to a known sample or investigated for specific alleles

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A B C D E F G

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EcoEco

Eco DigestEco DigestEco cuts to yield two DNA fragments

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EcoEco

EcoEco

BglBgl

BglBgl

OrOr

Bgl DigestBgl Digest

Bgl also cuts to yield two DNA fragments. But where is the Bgl site in relation to the Eco site?

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EcoEco BglBgl

EcoEco Bgl Bgl DoubleDouble Digest Digest

Shows it must be: Shows it must be:

A restriction digest with both Eco and Bgl enzymes provides the answer.

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Review• Restriction enzymes cut up DNA into pieces unique to

your DNA– DNA pieces are a function of your genetics and the

restriction enzyme used• DNA fragment soup placed in Gel well and distributes

based on fragment/sequence length• Resulting gel is unique—like a fingerprint

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Your Turn:

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

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WHAT ARE RE’S USED FOR?

Genetic engineering – pasting together DNA from two different organisms.

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

STICKY ENDS

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HOW IS DNA PASTED TOGETHER?

Ligase – another enzyme which reconnects phosphodiester bonds.

RE Video

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Videos 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

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WHAT ELSE ARE RE’S USED FOR?

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

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

How frequently would EcoRI cut DNA?

46= once every 4096 bp

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

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THOUGHT QUESTION

Bacteria are prokaryotes.

Prokaryotes do not have a nucleus.

Both DNA and RE’s are in cytoplasm.

Why isn’t bacterial DNA cut by RE’s?

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Methylation

See board

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

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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.

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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

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• Different restriction enzymes cut DNA in different ways.

– each enzyme has a different restriction site

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– some cut straight across and leave “blunt ends”

– some make staggered cuts and leave “sticky ends”

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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.

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

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

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• 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