DNA Technology

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14 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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DNA Technology


In laboratory experiments


Genes can be transcribed and translated after
being transplanted from one species to another



Called “Recombinant
DNA” technology



Can be produced via
“Genetic Engineering”
(laboratory manipulation)

1

Overview: Understanding and Manipulating Genomes


One of the greatest achievements of
modern science has been the
sequencing
of the human genome
, which was largely
completed by 2003


DNA sequencing accomplishments


Have all depended on advances in DNA
technology, starting with the invention of
methods for making recombinant DNA


DNA sequencing
animation

2

How can we modify a person’s
genome?


Gene therapy
-

insertion of genetic material
into human cells to treat a disorder



Ex vivo therapy


cells are removed for a person
altered and then returned to the patient



In vivo therapy


a gene is directly inserted into an
individual through a vector (e.g. viruses) or directly
injected to replace mutated genes or to restore
normal controls over gene activity



Gene therapy has been most successful in
treating cancer, to date.


Genomics

Ex vivo gene therapy

Genomics

defective gene

retrovirus

normal gene

normal gene

viral recombinant RNA

viral recombinant DNA

1. Remove bone


marrow stem cells.

2. Use retroviruses


to bring the normal


gene into the bone


marrow stem cells.

4. Return genetically


engineered cells


to patient.

3. Viral recombinant


DNA carries normal


gene into genome.

viral recombinant

RNA

reverse transcription

Copyright © The McGraw
-
Hill Companies, Inc. Permission required for reproduction or display.

DNA Cloning


Concept 20.1: DNA cloning permits
production of multiple copies of a specific
gene or other DNA segment


To work directly with specific genes


Scientists have developed methods for
preparing well
-
defined, gene
-
sized pieces of
DNA in multiple identical copies, a process
called gene cloning

5

24.3 DNA Technology


The Cloning of a Gene


Cloning:



Production of many identical copies of




an organism through some asexual means.



Gene Cloning:

The production of many identical





copies of a single gene



Two Ways to Clone a Gene:


Recombinant DNA


Polymerase Chain Reaction

6

Using Restriction Enzymes to Make Recombinant DNA


Bacterial
restriction
enzymes


Cut DNA
molecules at
a limited number of
specific DNA
sequences
, called
restriction sites

7

Restriction Enzymes and Sticky Ends

Step through animation

of cut/splice using EcoRI


Narrated animation

8

Cloning of a Human Gene / Recombinant DNA


Restriction enzymes breaks
open a plasmid vector at
specific sequence of bases
“sticky ends”


Foreign DNA that is to be
inserted is also cleaved with
same restriction enzyme so
ends match


Foreign DNA is inserted into
plasmid DNA and “sticky ends”
pair up


DNA
ligase

seals them together


Narrated animation

of “Cloning
a Gene”

9

24.3 DNA Technology


Polymerase Chain Reaction


Amplifies a targeted DNA
sequence


Requires DNA polymerase, a set of
primers
, and a supply of
nucleotides


Primers are single stranded DNA
sequences that start replication
process


Amount of DNA doubles with each
replication cycle


Process is now automated


Narrated animation



Step by step animation

10

24.3 DNA Technology


DNA Fingerprinting


Permits identification of individuals and their relatives


Based on,

polymorphisms

differences between
sequences in nucleotides between individuals


RFLP
s : restriction fragment length polymorphisms


Narrated animation


Detection of the number of repeating segments (called
repeats) are present at specific locations in DNA


Different numbers in different people


PCR amplifies only particular portions of the DNA


Procedure is performed at several locations to identify repeats

11

DNA Fingerprints

DNA fragments (after digest with restriction
enzymes) can be separated through

gel
ELECTROPHORESIS


See How:

Animation
http://highered.mcgraw
-
hill.com/sites/007337797x/student_view0/cha
pter14/animation_quiz_
-
_dna_fingerprinting.html



Step
-
by
-
step electrophoresis



Another
walk
-
through explanation

12

Forensic Evidence


DNA “fingerprints”
obtained by analysis of
tissue or body fluids
found at crime scenes


Can provide definitive
evidence that a suspect is
guilty or, more specifically,
not guilty


Is a specific pattern of
bands of RFLP markers
on a gel

Defendant’s

blood (D)

Blood from

defendant’s

clothes

Victim’s

blood (V)

D

Jeans

shirt

V

4
g

8
g

Figure 20.17

13

DNA fingerprinting

Can also be used in
establishing paternity


Figure
: Electrophoresis of PCR
-
amplified DNA fragments. (1)
Father. (2) Child. (3) Mother. The
child has inherited some, but not all
of the fingerprint of each of its
parents, giving it a new, unique
fingerprint.

http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Paternity_testing

14

24.3 DNA Technology


Biotechnology



Biotechnology uses natural biological systems to
create a product or to achieve a goal desired by
humans.




Model Organisms
” favored for genetics research


15

Environmental Cleanup


Genetic engineering can be used to modify
the metabolism of microorganisms


So that they can be used to extract minerals
from the environment or degrade various types
of potentially toxic waste materials

16

24.3 DNA Technology


Transgenic Bacteria



Medical Uses: Production of Insulin, Human Growth
Hormone, Hepatitis B Vaccine



Agricultural Uses: Bacteria that protects plants from
freezing, bacteria that protect plant roots from insects



Environmental: Bacteria that degrade oil (clean up
after oil spills), bacteria that remove sulfur from coal


17

24.3 DNA Technology


Transgenic (GM) Plants


Plants have been engineered to secrete a toxin that
kills insects (ex: Bt corn)



Plants have been engineered to be resistant to
herbicides (ex: Roundup Ready)



Animation: Gene Transfer in Plants Using a Ti Plasmid
http://glencoe.mcgraw
-
hill.com/sites/9834092339/student_view0/chapter17/genes_into_plants_
using_the_ti
-
plasmid.html



18

Agricultural Applications


DNA technology


Is being used to improve agricultural
productivity and food quality

19

In 2008:

92% of the
soybeans and
80% of the
corn planted in
the United
States had
been
genetically
engineered.


Genetic Engineering in Plants


Agricultural scientists


Have already endowed a
number of crop plants with
genes for desirable traits

Bt corn (right)

20

Biotechnology products:

Transgenic plants

DNA technology

Copyright © The McGraw
-
Hill Companies, Inc.
Permission

required for reproduction or display.

b. Salt
-
intolerant Salt
-
tolerant

a. Desirable traits

Disease
-
protected

Wheat, corn, potatoes

Herbicide
-
resistant

Salt
-
tolerant

Drought
-
tolerant

Cold
-
tolerant

Improved yield

Modified wood pulp

Wheat, rice, sugar beets, canola

Cereals, rice, sugarcane

Cereals, rice, sugarcane

Cereals, rice, sugarcane

Cereals, rice, corn, cotton

Fatty acid/oil content

Protein/starch content

Amino acid content

Corn, soybeans

Cereals, potatoes, soybeans, rice, corn

Corn, soybeans

Transgenic Crops of the Future

Improved Agricultural Traits

Improved Food Quality Traits

Trees

Health focus: Ecological concern about
GMO crops


Resistance increasing in the target pest



Exchange of genetic material between the
transgenic plant and a related species



Concern about the impact of BT crops on
nontarget species (ex: pollinators)

DNA technology

GM Animals and “Pharm”
Animals


Transgenic animals


Contain genes from other organisms


Sometimes called “chimeras”


Fig 1. transgenic mouse lines expressing
GFP

known as “green
mice.”


23

24.3 DNA Technology


Transgenic
Animals


Fish, cows, pigs,
rabbits and sheep
have been
engineered to
produce human
growth hormone in
order to increase
size of the animals


24


Transgenic organisms
have a foreign gene
inserted into their DNA


Have been engineered to be pharmaceutical
“factories”

Figure 20.18

“Pharm” Animals

25

Human breast milk from a cow
(2011)

Pharmaceutical Products

26


“Knockout” mice

A number of mice models have been developed: either possessing an
inactivated tumor suppressor gene (p53), an activated oncogene (Tg.AC),
over
-
expression of a (human) oncogene (rasH2) or being deficient in
nucleotide excision repair (Xpa, de Vries et al., 1995).


These mice models have several advantages:


the number of animals needed for one study is 120 instead of 400
-
500


the duration of the study is 6
-
9 instead of 24 months leading to less
distress of the animals


the transgenic mouse model is considered more discriminating hence
improving the accuracy and reliability of human carcinogen identification.

http://www.nca
-
nl.org/English/Newsletters/Nb13/nl13txt.html

27

Safety and Ethical Questions Raised by DNA Technology


The potential benefits of genetic engineering


Must be carefully weighed against the potential
hazards of creating products or developing
procedures that are harmful to humans or the
environment


Today, much public concern about possible
hazards


Centers on genetically modified (GMOs)
organisms used as food (allergic reactions,
etc)


Gene “escape”

28