Download 3 Intro to Restriction Enzymes

Biotechnology
The use of biological processes,
organisms, or systems to
manufacture products intended to
improve the quality of human life.
Applications of Biotechnology
•
•
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Industry
Medicine
Agriculture
Forensics
What we will cover…
• Transgenic Organisms- ex GMO’s
– Contain recombinant DNA
• Gene Therapy
• Cloning– organisms OR genes (PCR)
• Gel Electrophoresis
• Stem Cells
What are Restriction Enzymes?
• Proteins
• In nature: Used by bacteria to cut viral
DNA
• They “restrict” the growth of viruses
• http://www.dnalc.org/resources/animatio
ns/restriction.html
Restriction Enzymes
• Cut strands of DNA at specific nucleotide
sequences
• There are many different restriction
enzymes that each cut DNA at different
nucleotide sequences
• Usually occurs at a palindrome
5’ GAATTC
3’ CTTAAG
What is a palindrome in
English?
• Palindromes can be read backwards and
forwards the same way.
• Give me an example of a name that is a
palindrome:
– Hannah
• It’s a little more tricky in DNA because the
strand itself isn’t what must be backwards
and forwards…
DNA palindromes
• A DNA palindrome reads exactly opposite
its complementary strand
• Ex- CAGAC is not a palindrome but
CCCGGG is. Why?
• Let’s look at the complementary strand:
• CAGAC
CCCGGG
• GUCUG
GGGCCC
Another Example
Answers to worksheet
• 5’ C T G C A G 3’
3’ G A C G T C 5’
• 5’ A G C C G A 3’
3’ T C G G C T5’
• 5’ G T T A A C 3’
3’ C A A T T G 5’
25%
25%
25%
25%
Which of these pieces of DNA
represents a palindrome?
1. 5’ TTTCGC 3’
3’ AAACCG 5’
2. 5’ GAATTC 3’
5’ CTTAAG 3’
3. 5’ GAATTC 3’
3’CTTAAG 5’
4. 5’ AATGCC 3’
5’ TTACGG 3’
Sticky Ends
• Most restriction enzymes cut
DNA with a staggered cut
• The staggered cuts leave
the DNA with end pieces
“sticking off”
– We call these “sticky ends”
– These exposed N-bases will
want to join with other
complimentary exposed
bases
Sticky vs Blunt Ends
• If a restriction enzyme
cuts to make blunt
ends, there aren’t any
nitrogen bases
hanging off to join
together with another
piece of DNA.
So what ?
• What do you predict could happen if two
pieces of DNA are cut with the same
restriction enzyme???
– YES! They will have the same “sticky ends”
– How could we use this???
Recombinant DNA
• Recombinant DNA contains DNA from
2 or more different sources.
• So how does DNA get from one species
to another?
• Vector: something used to transfer DNA
from one organism to another
– Microinjection
– Gene Gun
– Virus
– Bacteria Plasmid
Transgenics
• Genetic engineering
makes it possible to
transfer DNA sequences
from one organism to
another
• REMEMBER- DNA is
universal
• A gene taken from one
species can be
replicated in another
species.
• So how do I take a gene from one
organism and put it into another?
• We have to cut it using a Restriction
Enzyme
Bacteria are very
useful
• Bacteria are used because they have
plasmids
• A plasmid is a circular
piece of DNA that exists
apart from the chromosome
and replicates independently
of it.
Restriction Enzymes can be used to make
RECOMBINANT DNA!
• The gene you are interested in inserting
(aka the “gene of interest”) can be cut
using a restriction enzyme.
• What will happen if I also cut the other
organisms DNA with the same Restriciton
Enzyme?
Making Recombinant DNA…
• Once the gene is isolated, how do we join
it with the organism’s DNA?
• Cut the organism’s DNA with the same
restriction enzyme…why
– The sticky ends will naturally be attracted to
each other
• Add DNA LIGASE: an enzyme that seals
the fragments together
• Restriction Enzymes can be used to make
Recombinant DNA.
• The gene of interest can be isolated using
Restriction Enzymes
Applications of this technology
• PCR
• GMO’s- (Transgenic Organisms)
• Gene Therapy
PCR
• Now whenever the bacteria plasmid
replicates, the gene will be cloned.
• Gene Cloning: the production of many
identical copies of the same gene.
• You have created a
Transgenic Organism!
–organisms that contain functional
recombinant DNA (rDNA) from a
different organism
Gene Therapy
• Cystic Fibrosis example
• http://learn.genetics.utah.edu/content/tech/
genetherapy/cysticfibrosis/
The Human Genome
• A map of all the base pairs in human DNA.
• Video- alopecia and the Human Genome
Project