Biotechnology

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21 Φεβ 2013 (πριν από 4 χρόνια και 7 μήνες)

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Biotechnology

Manipulation of Life for
Knowledge and Application

What is Biotechnology

The broad definition of biotechnology is simply
the industrial use of living organisms

(or
parts of living organisms)
to produce foods,
drugs, or other products
.

The oldest biotechnologies include
fermentation and plant and animal
hybridization
.

The newest biotechnologies range from
protein separation technologies to genomics
and combinational chemistry.


A sampler of fields that fall under biotechnology's broad
umbrella would include:


bacteriology


biochemical engineering


bioinformatics


bioprocessing


cell biology


chromatography


computational &
mathematical modeling


developmental and
molecular genetics


DNA


technologies


electrophoresis


embryology


immunology


materials science


microbiology


nucleic acid chemistry


protein engineering


virology


Areas of Focus

Procedures


electrophoresis, DNA extraction, Gene
Isolation, PCR, Transformation

Applications


DNA profiling, genetic screening,
engineered crops, pharmaceutical
production, cloning

Ethics


Just because we can, does not mean we
should



Procedures

Extraction


Purification of DNA or mRNA from cells

Restriction Digest


Cutting DNA into fragments with enzymes

Gel Electrophoresis


Separation of DNA or RNA fragments based on length

Polymerase Chain Reaction


PCR


Amplification of DNA or RNA

Transformation


Placement of DNA fragment into vector and insertion into cell

The Big Picture

Several procedures
are used together to
create a final
product

Gel electrophoresis

Principle:

1.
DNA and RNA have an overall negative
charge due to the phosphate groups.

2.
If placed in a electric field the nucleic acids
will move toward the positive pole of the
field.


3.
In a gel matrix, smaller fragments will move
faster than larger fragments.

4.
The DNA or RNA is separated by the length
of the molecules

DNA and RNA have an overall negative charge due
to the phosphate groups.

If placed in an electric field the nucleic acids will move
toward the positive pole of the field

In a gel matrix, smaller fragments will move faster than
larger fragments

The DNA or RNA is separated by the length of
the molecules

Gel Electrophoresis Animation

Extraction

Principle:


Removal and purification of genetic
material from living or dead cells


Uses chemical and mechanical means


Extracted DNA can be sequenced,
screened, or amplified


Identified genes can be inserted into
genome of new organism

DNA Extraction

Procedure


Lyse (break open) cells by
grinding or sonication, add
detergent to break up
membranes


Precipitate proteins with sodium
(salt) or degrade proteins with
protease enzyme


Precipitate DNA with cold
ethanol or isopropyl alcohol.

Restriction Digest

Principle:


Uses restriction enzymes to cut
strands of DNA at specific sequences


Can be used to create a DNA
fingerprint or isolate specific genes


Also used to cut open circular DNA
such as plasmids

Restriction Enzyme

A protein that
recognizes a
specific DNA
sequence and
severs the double
helix.

Hundreds of different
enzymes have been
isolated and can be
purchased

for lab
use

Each enzyme recognizes and cuts a different
DNA sequence

Restriction Digest Reaction

To a tube add…


Sample DNA


Restriction enzymes


Deionized water


Buffer solution (ph, salt
specific to enzyme)

Incubate at 37
°
C for 2
hours to overnight



Cut DNA can now
be…


separated using
electrophoresis to
generate a DNA
fingerprint


amplified using PCR


ligated (combined)
with other DNA
fragments in the
case of plasmids

Polymerase Chain Reaction
-

PCR

Principle:

Specific sequences of DNA can be
amplified in a short period of time.

From a single copy of a gene more than
a million can be synthesized in a few
hours
.

Amplified DNA fragments can be ligated
into plasmids, or sequenced


The Taq DNA polymerase

PCR uses the special
properties of a DNA
replicating protein
discovered in
Thermus aquaticus


The Taq polymerase
is only active at
72
°
C

It can amplify 1kb of
DNA is 30 seconds

The Cycling Reactions

There are three major steps in
a PCR, which are
repeated for 30 or 40
cycles.


1.
Denaturation:
separating
double helix

2.
Annealing:
short DNA
primers bind

3.
Elongation:
DNA is
replicated, beginning at
primer location



This is done on an automated
cycler, which can heat
and cool the tubes with
the reaction mixture in a
very short time

PCR works exponentially.. 2, 4, 8, 16, 32, 64…

PCR Video

Transformation

Principle:


Digested or amplified DNA is inserted
into a plasmid


Cells are induced to uptake plasmids


Within cells the inserted genes are
expressed


Resulting proteins will affect
phenotype of organism

Plasmids

Plasmids are small rings of DNA, usually
created by molecular biologists to have
specific characteristics such as origins of
replication, multiple restriction sites, and
antibiotic gene.

Ligation

1.
Plasmid and
selected DNA
fragment are cut
with same
restriction enzyme

2.
Incubated together
with DNA ligase

3.
DNA is inserted
into plasmid


Tranformation

Cells are induced
to uptake
plasmid

The gene can now
be expressed,
conferring its
trait upon the
cell

Creating a DNA Library
-

Animation

Applications

DNA Profiling


Crime scene investigation, paternity cases

Genetically Modified Food


Pesticide resistant corn, vitamin enriched rice,

Cloning


Creating genetically identical organisms

Genetic Screening


Predicting future health issues by looking at genes

Pharmaceutical Production


Production of insulin, antibodies, and other life enhancing
molecules

Ethics

“To what degree should we meddle with
the power of life, a power which we
know so very little of, but which we are
entirely dependent upon?”






-

some guy

“One day we will unlock the door that
leads to the body immortal, but will you
step through it?”






-

the same guy