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
Genomics
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
2. Use retroviruses
to bring the normal
gene into the bone
marrow stem cells.
1. Remove bone
marrow stem cells.
retrovirus
defective gene
4. Return genetically
engineered cells
to patient.
viral recombinant DNA
reverse transcription
viral recombinant RNA
3. Viral recombinant
DNA carries normal
gene into genome.
normal gene
viral recombinant
RNA
normal gene
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
– RFLPs : 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.mcgrawhill.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
Defendant’s
blood (D)
Blood from
defendant’s
clothes
4
D
Jeans
g
8
shirt
Victim’s
blood (V)
g
V
– 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
Figure 20.17
13
DNA fingerprinting
Can also be used in
establishing paternity
Figure: Electrophoresis of PCRamplified 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.mcgrawhill.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
In 2008:
92% of the
soybeans and
80% of the
corn planted in
the United
States had
been
genetically
engineered.
19
Genetic Engineering in Plants
• Agricultural scientists
– Have already endowed a
number of crop plants with
genes for desirable traits
Bt corn (right)
20
DNA technology
Biotechnology products:
Transgenic plants
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Transgenic Crops of the Future
Improved Agricultural Traits
Disease-protected
Wheat, corn, potatoes
Herbicide-resistant
Wheat, rice, sugar beets, canola
Salt-tolerant
Cereals, rice, sugarcane
Drought-tolerant
Cereals, rice, sugarcane
Cold-tolerant
Cereals, rice, sugarcane
Improved yield
Cereals, rice, corn, cotton
Modified wood pulp
Trees
Improved Food Quality Traits
Fatty acid/oil content
Corn, soybeans
Protein/starch content Cereals, potatoes, soybeans, rice, corn
Amino acid content
a. Desirable traits
Corn, soybeans
b. Salt-intolerant
Salt-tolerant
DNA technology
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)
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”
“Pharm” Animals
Figure 20.18
Human breast milk from a cow
(2011)
25
Pharmaceutical Products
26
• “Knockout” mice
http://www.nca-nl.org/English/Newsletters/Nb13/nl13txt.html
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. 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