Biotechnology

mutebabiesΒιοτεχνολογία

6 Δεκ 2012 (πριν από 5 χρόνια και 1 μήνα)

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Biotechnology

Use of living things to provide
needed products or processes

Recombinant DNA


DNA produced by joining
segments of DNA from different
sources



eg. To produce human insulin,
scientists have combined bacterial
plasmid DNA + human DNA

Tools for Producing
Recombinant DNA

Restriction enzymes: enzymes that
cleave the DNA double helix at
specific nucleotide sequences


Use of the Restriction Enzyme
Bam H1


5’


G G A T C C


3’


3’


C C T A G G


5’



5’


G G A T C C


3’


3’


C C T A G G


5’


sticky end

sticky end

Results in

Tools for Producing
Recombinant DNA

Vector: carrier of DNA; can be virus or plasmid


Plasmid: extrachromosomal, independently
replicating, small circular DNA molecule

Producing Recombinant DNA


restriction

enzyme

Treat source
DNA with
restriction

enzyme


Treat plasmid
DNA with
same enzyme

restriction

enzyme

Mix together

Add DNA Ligase

Many recombinant DNA

molecules are produced,

each with a different
piece of source DNA

Transform


bacterial cells

Each bacterial cell

carries a different

recombinant plasmid

Tools for Producing
Recombinant DNA

Probe: sequence of DNA that is
complementary to the gene of interest;
Used to locate a copy of the gene by
hybridization

Add Probe

Probe Binds to gene

AGCTTAGCGAT

TCGAATCGCTA

AATCGC

AGCTTAGCGAT

TCGAATCGCTA

Denature DNA by heating

Building
a

DNA
Library

Finding the Gene of Interest

by Screening a Gene Library

Applying Your Knowledge

Applying Your Knowledge

A.
An enzyme that cleaves DNA at specific
sequences is a __________ .

B.
A sequence of DNA that is complementary to
the gene of interest is a _________.

C.
A small, independently replicating DNA
molecule is a ___________.

1.
Probe

2.
Clone

3.
Plasmid

4.
Restriction Enzyme

Biotechnological Methods: PCR

Polymerase Chain Reaction (PCR)




Amplifies a specific region in the DNA



Used for identification, especially


if the amount of DNA is small



Uses repeated cycles of heating to


denature DNA and cooling to synthesize


new DNA


Involves the use of


---
Taq polymerase (withstands heat)


---
primers to begin synthesis

Polymerase Chain Reaction:

One PCR Cycle

Original

Double
-

helix

DNA

Separate

DNA

Strands

90
°
C

Primers &

Taq

polymerase

bind

50
°
C

Taq Polymerase

Primer

72
°
C

DNA

synthesized

Polymerase Chain Reaction:

Multiple PCR Cycles

DNA

fragment

to be

amplified

2 copies

4 copies

8 copies

Biotechnological Methods: RFLP

RFLP Analysis


R
estriction
F
ragment
L
ength
P
olymorphism




Use of a probe to identify specific DNA


fragments derived from restriction enzyme


digestion



Shows variations in sizes of fragments


between different individuals



Separation of Restriction Fragments by Size


DNA separated by size

is transferred from

agarose gel to filter

DNA on filter is

exposed to probe

to detect

complementary

sequences.


Southern Blotting for RFLP Analysis

Applications of Biotechnology


in Agriculture


Transgenic: organism that contains a
gene from another species in all of its
cells



Transgenic plants that


Resist herbicides


Resist pests


Have improved storage qualities


Have enhanced nutrition


Roundup Ready Soybeans

Traditional Soybeans

Effects of Treatment with the Herbicide Roundup

Bt Corn: Produces

its own Pesticide

Flavr
-
Savr Tomato

softens more slowly

after ripening

“Golden” rice with
beta
-
carotene and
extra iron

Applications of Biotechnology


in Agriculture





Transgenic Animals that


Provide models for human diseases


Mice with BRCA1 gene to study


inherited breast cancer


“knockout” mouse missing ADA


gene to study immune deficiency


Produce pharmaceuticals and


release them in milk


Goats producing TPA, tissue
plasminogen activator, for treatment of
heart attacks

C

S

R

C

I

E

M

N

E

E

Applications of Biotechnology

for Identification: Forensics

1

2

3

4

5

6

7

Suspects

Suspects

Applications of Biotechnology

for Identification: Paternity Testing

X

X

X

X

X

X

X

X

Applications of Biotechnology

in Medicine: Therapeutics

Pharmaceutical

Used for

Factor VIII

Blood Clotting

Human Growth
Hormone

Pituitary
Dwarfism

Insulin

Diabetes

Interferon

Cancer

Vaccine

Hepatitis B

Applications of Biotechnology

in Medicine: Genetic Testing

carrier

sickle
-
cell anemia

carrier

non
-
carrier

Applications of Biotechnology

in Medicine: Gene Therapy


Andrew Gobea was diagnosed with ADA deficiency


before he was born.


This lack of the ADA enzyme causes an immune


deficiency disease called SCID.


Andrew was given the gene for a functional ADA


enzyme four days after his birth in 1993.


Umbilical cord blood was collected at birth.


Stem cells were isolated and mixed with a virus


carrying a functional ADA gene.


Stem cells were returned to Andrew with the aim of


populating his bone marrow with cells that could


make the ADA enzyme.

Two Years Later


Andrew has been maintained on costly injections of


purified ADA enzyme while waiting to see if the


gene therapy has been effective.


Tests have shown that the functional ADA gene


was introduced into 0.01% of Andrew’s stem cells


and this population has given rise to 5
-
7% of his


white blood cells. Is this enough for a cure?

Four Years Later


Andrew’s physician decides to taper off the enzyme


injections to see if Andrew’s immune system can


protect him.



What is the outcome?




What was learned?