Biotechnology is the use of biological processes, organisms, or systems to

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22 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

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Biotechnology is the use of biological
processes, organisms, or systems to
manufacture products intended to improve
the quality of human life.

Genetic Engineering
-

(A.K.A. Recombinant
DNA Technology)




frequency of an allele in a
population


*involves cutting (cleaving) DNA
from one organism into small
fragments & inserting the
fragments into a host organism
of the
same

or a
different

species


AMAZING!!!


Organism will use the foreign DNA as
if it were its own!!


Transgenic Organism
-

organisms that contain
functional recombinant DNA (rDNA) from a different
organism


4 Areas of Biotechnology


Agriculture



Industry




Forensics


Medicine


Remember DNA?


What is the monomer of DNA?


Nucleotides


How do bases pair?


A


T


C


G


What kind of bond is used?


Hydrogen bonds between nitrogen bases




I. Restriction Enzymes


AKA Restriction
Endonucleases


What macromolecule do
you think they are made
of?


They are PROTEINS that cut
strands of DNA at specific
nucleotide sequences

Isolating foreign DNA fragments



-
Restriction Enzymes
-

DNA cutting enzymes that
can cut both strands of a DNA molecule at a specific
base pair sequence (A
-
T, C
-
G)


-
similar to cutting a zipper into pieces


-
must find the same sequence of base pairs on
both DNA strands but they must run in opposite
directions


Restriction Enzymes (cont.)

A.
There are many different restriction enzymes that each
cut DNA at different nucleotide sequences

B.
Most will cut the DNA with a staggered cut

C.
Usually occurs at a
palindrome
: a sequence of units
that can be read the same way in either direction (ex.
Mom, dad, racecar)


5‘…GAATTC…3’

3‘…CTTAAG…5’

Action of Restriction Enzymes



Sticky Ends

1.
The staggered cuts leave the DNA with end pieces
“sticking off”

a.
We call these “sticky ends”

b.
These exposed N
-
bases will want to join with other
complimentary exposed bases

E. Types of Restriction Enzymes

1.
Sticky End
-

already discussed

2.
Blunt End

a.
These cut the DNA straight across and create blunt
ends:



CCC GGG

GGG CCC

F. Products generated by restriction
enzymes

1. COHESIVE END CUTTERS (staggered cuts):


Enzyme



Recognition Site




Ends of DNA After Cut


2. BLUNT END CUTTERS (direct cuts):


Enzyme



Recognition Site



Ends of DNA After Cut

5’…GAATTC…
3’

3’…CTTAAG…
5’

5’…G


AATT
C…3’

3’…C
TTAA


G…5’

Eco
RI

Pst
I

5’…CTGCAG…3


3’…GACGTC…5


5’…C
TGCA


G…3’

3’…G



ACGT
C…5’

Hae
III

5’…GGCC…
3’

3’…CCGG…
5’

5’…GG


CC…3’

3’…CC


GG…5’

1. Restriction enzymes are named according to the
following nomenclature:

Ex: EcoRI



E = genus Escherichia


co = species coli


R = strain RY13


I = first enzyme isolated


G. Restriction Enzyme Naming

How is a transgenic organism formed??



Isolate foreign DNA fragment


Attach DNA fragment to a “vehicle” (vector)


Transfer “vehicle” (vector) into a host organism


Forming transgenic organisms and therefore clones of
genes

Why would anyone go through the trouble
of cutting DNA???


One reason…


Recombinant DNA


Break down the word…what do you think recombinant
means?


Other reasons…


DNA fingerprinting, gene therapy…


Recombinant DNA

A.
Recombinant DNA: DNA that has been cut from
one strand of DNA and then inserted into the gap of
another piece of DNA that has been broken.

1.
The host DNA is often a bacterial cell such as
E coli
.

Bacterial Structure

1.
Bacteria are often used in biotechnology
because they have plasmids

2.
A
PLASMID
is a circular piece of DNA that
exists apart from the chromosome and replicates
independently of it.

3.
A plasmid is therefore called a
VECTOR
.


Vectors transfer DNA



Vector
-
means by which DNA from
another species can be carried into the
host cell


Mechanical Vectors


Micropipette
-
inserts into a cell


Gene guns
-

tiny metal bullet is coated
with DNA and shot into the cell with a
gene gun


More types of Vectors


Biological Vectors


Viruses


Plasmids
-
small ring of DNA found in bacteria
cells that is separate from the bacteria’s
normal set of DNA


Plasmid usually contains genes that may cause the
bacteria to be resistant to certain antibiotics

D. Isolating Genes

1.
Must isolate the gene
of interest first before
you insert it into the
plasmid

2.
How do you do this?

a.
Use a restriction
enzyme!!!


Final Steps of Making Recombinant DNA

1.
Once the gene is isolated, have to cut the organism’s DNA
with the same restriction enzyme…why?

a.
The sticky ends will naturally be attracted to each other

2.
Add DNA LIGASE: enzyme that seals the fragments
together

3.
After the foreign DNA has been spliced (glued) into the
plasmid using an enzyme DNA ligase, the rDNA is
transferred into a bacterial cell or other organism

4.
Now organism is called a
Transgenic Organism
-

organisms that contain functional recombinant DNA
(rDNA) from a different organism






Gene Splicing/Cloning using a bacterial
plasmid



-
IMPORTANT


plasmid replicates separately from the
bacterial chromosome & can produce up to 500 copies
per bacterial cell


-
bacteria reproduce quickly (20 min) so a lot of rDNA is
made very fast


You will essentially be
cloning a gene
-

genetically
identical copies of rDNA molecules


-
Host cell produces the protein coded for by the rDNA

III. Uses for Recombinant DNA

A.
Recombinant DNA has been gaining importance over the
last few years, and will become more important as genetic
diseases become more prevalent and agricultural area is
reduced. Below are some of the areas where Recombinant
DNA will have an impact:


1.
Better Crops (drought & heat resistance)

2.
GMO’s (crops like seedless watermelon, pluots, etc.)

3.
Recombinant Vaccines (i.e. Hepatitis B)

4.
Production of clotting factors

5.
Production of insulin

6.
Production of recombinant pharmaceuticals

7.
Plants that produce their own insecticides

8.
Germ line and somatic gene therapy


RECAP


Steps for making a
transgenic organism:

1.
Locate and isolate the
gene of interest

2.
Cut out the gene and cut
the plasmid using the
appropriate restriction
enzyme

3. Insert the desired gene into the plasmid matching up
the sticky ends



4. Use the enzyme DNA ligase to seal up
the sticky ends

5. Transfer the vector in the host organism where it will
replicate

6. Host organism produces the protein coded for by the
recombinant DNA


Insulin Production

Cloning a gene


Transgenic Animals


Cloning an animal


Plants have been genetically modified to
produce insect toxin


Gene Therapy


Gene therapy attempts to treat genetic diseases at
the molecular level by correcting what is wrong
with defective
genes
.


Clinical research into gene therapy’s safety and
effectiveness has just begun.


No one knows if gene therapy will work, or for
what diseases.
If gene therapy is successful, it
could work by preventing a protein from doing
something that causes harm, restoring the
normal function of a protein, giving proteins
new functions, or enhancing the existing
functions of proteins

Gene Therapy


In vivo gene therapy

requires that the gene transfer
vector be delivered by
direct tissue injection.


2)
Ex
-
vivo gene therapy

involves
removing tissue
from the patient
, transfecting (or virally
-
infecting)
the cells in culture, and then reimplanting the
genetically altered cells to the patient.


Ex vivo gene therapy


In Vivo Gene Therapy