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BIOTECHNOLOGY
-intentional manipulation of genetic material of an organism
•
Deoxyribonucleic Acid (DNA)
• determines the characteristics
of all living organisms.
• occurs in most cells of all
organisms
• composed of four different
nucleotides in different
combinations
• each cell in the human body
contains more than 3 BILLION
letters
Four bases:
Adenine
Thymine
Guanine
Cytosine
2 bonds
3 bonds
• Sugar and phosphate backbone
• Double helix structure
(two spirals around each other)
• The only difference between living
organisms is the amount and order of
the four nucleotide bases.
Genome: the entire sequence of DNA
Gene: the part of the code that
corresponds to a protein
*genes can be transferred from one
organism to another*
BIOTECHNOLOGY
The intentional manipulation of genetic material of an organism
Why would we want to do this?
• To study cellular processes of an organism
– E.g. Glowing gene from jellyfish to tobacco plant
• To give one organism the trait(s) of another
– E.g. Anti-freeze from fish blood into
strawberries to survive through early frosts
Part 1: Manipulating Bacteria:
The Making of a Plasmid
Plasmid:
- a small circular piece
of extra-chromosomal
bacterial DNA, able to
replicate
- bacteria exchange
these plasmids to share
DNA
- E.g. antibiotic
resistance genes
• Since plasmid is made of DNA it can code
for genes, ex. antibiotic resistance, and
can carry specific sequences of DNA
• Specific DNA
sequences can be
recognized by
enzymes called
restriction
endonucleases
Restriction Endonucleases/Restriction Enzymes
• enzymes that are
able to cut doublestranded DNA into
fragments at
specific recognition
sites in DNA
sequences
Ex. EcoRI:
5’-GAATTC-3’
3’-CTTAAG-5’
• Restriction enzymes can create “sticky ends
or “blunt ends”
Sticky Ends
• fragment end of a
DNA molecule with a
short single-stranded
overhang
Blunt Ends
• fragment end of a
DNA molecule with
no overhang
Once made, the ends can be re-joined together by
other enzymes ("enzyme glue")
To Make a Recombinant Plasmid:
Insert
1. Cut the plasmid and the
insert with the same
restriction endonuclease
to make complementary
sticky ends.
2. Combine the sticky ends
using ligase.
ligase: enzyme used to
join DNA together
3. Introduce the
recombinant plasmid into
bacteria.
Making a Recombinant Plasmid
Bacterial Transformation
• introduction of foreign DNA into a bacterial cell
• plasmid is used as a vector, a vehicle by which DNA
can be introduced into host cell
----
---+
+
- ++ - ++
-+ -+-+- +- -+
------
plasmid
+++++
phospholipid
bilayer
- ++ -++ Ca2+ ions
Following transformation bacteria are grown in
medium with antibiotic…
Only the bacteria that have the plasmid (and
therefore the antibiotic resistance) will survive.
Example plasmid:
Origin of Replication:
• where the plasmid starts to duplicate itself
• the specific sequence MUST NOT be cut by restriction
endonucleases or it won’t be able to replicate
Part 2: Where do we get our insert sequence?
• From someone else’s DNA
– ex. fish gene in strawberries,
– jellyfish gene in plants
• Make it!
• In order to do these things, we need a way
to make many copies of the genes we want
Using Bacteria as Production Factories
•
•
•
•
easy to grow
no ethical issues
small genome
easy to manipulate
Making an insert:
Polymerase Chain Reaction
Common uses of biotechnology:
1. Making "stuff”
• proteins, enzymes, medication, etc. can be
produced by engineered bacteria!
• Food can be altered to have new traits
• Cloning (therapeutic and reproductive)
2. Genetic screening
• crime cases, relationship, genetic
screening, etc.
3. Gene Therapy
Therapeutic cloning
• used to produce tissue
that is identical to the
donor, to prevent
rejection
Reproductive Cloning
• creates an organism with the same
genetic material (DNA) as the
original organism – an EXACT
COPY of the donor
Dolly the Sheep
• the first cloned sheep
Ex. RFLP: Restriction Fragment Length
Polymorphism
Comparison of
different lengths
of DNA fragments
produced by
restriction
enzymes to
determine genetic
differences
between
individuals
Gene therapy
- desired gene is
inserted into cell's
nucleus using a
retrovirus as a carrier
- defective gene
replaced by functional
gene
Ex. ADA deficiency
- adenosine deaminase deficiency
- little immunity with low chances of recovery
- the T-cells of a four-year-old were
removed, modified and re-inserted to fix
her immune system