BIOTECHNOLOGY -intentional manipulation of genetic material of ...

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22 Οκτ 2013 (πριν από 3 χρόνια και 11 μήνες)

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BIOTECHNOLOGY

-
intentional manipulation of genetic material of an organism






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

D
eoxyribo
n
ucleic
A
cid (DNA)

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 double
-
stranded DNA into
fragments at
specific recognition
sites in DNA
sequences






Ex.
EcoR
I:




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:

1.
Cut the plasmid and the
insert with the same
restriction endonuclease
to make complementary
sticky ends.

Insert

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

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+ + + + +

-

++
-
++
-


-

++
-

++

+ + + + +

+
-

+

phospholipid
bilayer

plasmid

Ca
2+

ions



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

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
:



the specific sequence MUST NOT be cut by restriction


endonucleases or it won’t be able to replicate



where the plasmid starts to duplicate itself

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


easy to grow


no ethical issues


small genome


easy to manipulate

Using Bacteria as Production Factories



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