Download Genetic Engineering

Genetic Engineering
Biotechnology
2006-2007
Genetic Engineering
• Process of Manipulating & Using DNA
Genetic Engineering:
Isolating & Using Genes
• Manipulating DNA
– A.
• 1.
• 2.
• 3.
– B.
• 1.
• 2.
• 3.
The Tools of Molecular Biology
DNA Extraction
Cutting DNA
Separating DNA
Using the DNA Sequence
Reading the Sequence
Cutting and Pasting
Making Copies
Think About it…
• All living things have the same genetic
material (DNA) and use the same
genetic code.
• Gene sequences (instructions for
making a protein) can be “read” and
used in the same way by any and all
living things
• Biologists decided that, if given the
technology, genes for desirable traits
could be transferred from one
organism to another
The code is universal
• Since all living
organisms…
– use the same DNA
– use the same code
book
– read their genes the
same way
Can we mix genes from one
creature to another?
YES!
A Brave New World
How do we do mix genes?
• Genetic engineering
– find gene
– cut DNA in both organisms
– paste gene from one creature into other
creature’s DNA
– insert new chromosome into organism
– organism copies new gene as if it were its own
– organism reads gene as if it were its own
– organism produces NEW protein:
Remember: we all use the same genetic code!
Restriction Enzymes… “little
Molecular Scissors”
• Restriction enzymes
– “recognize” unique nitrogen-base sequences
– Called “recognition sites”
– different restriction enzymes “recognize”
different nitrogen-based sequences
– Enzymes “CUT” DNA strands at specific
locations
– DNA fragments now have uneven “sticky ends”
(unpaired bases)
– “Sticky ends” allow the DNA fragment to splice
(base –pair) into another open DNA strand
Cutting DNA
• DNA “scissors”
– enzymes that cut DNA
– restriction enzymes
• used by bacteria to cut up DNA of
attacking viruses
• EcoRI, HindIII, BamHI
– cut DNA at specific sites
• enzymes look for specific base sequences
GTAACGAATTCACGCTT
CATTGCTTAAGTGCGAA
Restriction Enzymes
Recognition
sequences
Original DNA strand
DNA
sequence
Restriction
enzyme EcoRI
cuts the DNA
into fragments.
Sticky end
Restriction enzyme cuts both strands of
the DNA Between the A and G following
a CTTAAG sequence
Figure 14-18 DNA Fingerprinting
Once the DNA is Cut, the “repeat” fragments are
Labeled
using
Section
14-3 radioactive probes allowing us to see bands
Following Electrophoresis
Restriction enzyme
Chromosomes contain
large amounts of DNA
called repeats that do
not code for proteins.
This DNA varies from
person to person.
Here, one sample has
12 repeats between
genes A and B, while
the second sample has
9 repeats.
Go to
Section:
Restriction enzymes
are used to cut the
DNA into fragments
containing genes and
repeats. Note that the
repeat fragments from
these two samples are
of different lengths.
The DNA fragments are
separated according to
size using gel
electrophoresis. The
fragments containing
repeats are then labeled
using radioactive
probes. This produces a
series of bands—the
DNA fingerprint.
Electrophoresis Lab
• /
Gel Electrophoresis used to separate the fragments by Size
And Charge
DNA plus
restriction
enzyme
Apply an Electric Field
Power
source
Longer
fragments
Mixture of
DNA
fragments
Gel
Shorter
fragments
An electric current causes the fragments to move from the well toward
The Positive end of the field; shorter fragments migrate farther and faster
Than the larger fragments
DNA fingerprint
• Genes form banding patterns
• No two banding patterns
are the same
• Called DNA fingerprints
– Used in identifying remains
– Crime investigations
– Next of kin
Figure
13-8isolated,
PCR
Once the genes
are
we can make
Extra
copies
through a cloning process
Section
13-2
Called PCR (polymerase Chain Reaction)
DNA polymerase adds
complementary strand
1st DNA heated to
separate strands
DNA fragment to
be copied
Go to
Section:
PCR
cycles 1
2
3
4
5 etc.
DNA
copies 1
2
4
8
16 etc.
Sneaking In
•You probably have heard of computer
viruses.
•Once inside a computer, these programs
follow their original instructions and
override instructions already in the host
computer.
•Scientists use small “packages” of DNA to
sneak a new gene into a cell, much as a
computer virus sneaks into a computer.
VECTORS …. Deliver the genes
• Vectors are used to deliver DNA
(genes) into another organism’s cells
• Vectors can be either:
– Plasmids (circular piece of bacterial DNA)
– Viruses “nonliving” particles that contain
a strand of DNA
“Prepping” the Vectors
• Recall that restriction enzymes are
used to cut-out the gene we want to
use
• “Sticky ends” are created (unpaired
bases)
• The SAME restriction enzyme is used
to cut and open-up the DNA of the
Plasmid or the Virus
• “Sticky ends” are created in vector’s
DNA too
Splicing the gene into the
Vector
• When the “sticky ends” of the vector
DNA comes into contact with the’
sticky ends of the selected gene;
they splice together!
• Now we have RECOMBINANT DNA
• Recombinant DNA is a product of
mixing the DNA of different
organisms
Bacterial DNA
• Single circular chromosome
– only one copy = haploid
– no nucleus
• Other DNA = plasmids!
bacteria
chromosome
plasmids
There’s more…
• Plasmids
– small extra circles of DNA
– carry extra genes that bacteria can use
– can be swapped between bacteria
• bacterial sex!!
• rapid evolution = antibiotic resistance
– can be picked up
from environment
How can plasmids help us?
• A way to get genes into bacteria easily
– insert new gene into plasmid
– insert plasmid into bacteria = vector
– bacteria now expresses new gene
• bacteria
make new protein
gene from
recombinant
other organism
cut DNA
plasmid
plasmid
+
vector
glue DNA
transformed
bacteria
Grow bacteria…make more
transformed
gene from
other organism
recombinant
plasmid
+
bacteria
vector
plasmid
grow
bacteria
harvest (purify)
protein
Splicing the gene we want into a Vector:
Making and Delivering Recombinant DNA
Recombinant
DNA
Cell with
Normal Gene
Gene for human
growth hormone
Gene for human
growth hormone
Human Cell
Sticky
ends
DNA
recombination
DNA
insertion
Bacterial Cell
Bacterial
chromosome
Plasmid
Using Bacteria as a Vector
Bacterial cell for
containing gene for
human growth
hormone
Figure 13-10 Plant Cell
Transformation
Using a Recombinant Bacteria Plasmid to insert desired gen
Into Plant
Agrobacterium
tumefaciens
Gene to be
transferred
Cellular
DNA
Recombinant
plasmid
Inside plant cell,
Agrobacterium
inserts part of its
DNA into host cell
chromosome
Plant cell
colonies
Transformed
bacteria introduce
plasmids into plant
cells
Complete plant is
generated from
transformed cell
Using a Virus as a Vector to insert Normal Hemoglobin
Gene into Bone Marrow cells
Gene Therapy
Bone
marrow
cell
Normal hemoglobin
gene
Chromosomes
Genetically
engineered virus
:
Nucleus
Bone
marrow
Viruses Normally infect cells by injecting their DNA,
So this process will occur naturally when viruses
Containing Recombinant DNA contact cells
Applications of Genetic Engineering
A.
Transgenic Organisms: contain recombinant DNA
1. Transgenic Microorganisms:
• Used as Vectors to carry desired gene to host
• Used to Produce Human Hormones (Insulin, Human Growth
Hormone)
2. Transgenic Animals: Contain DNA from another Organism
(Recombinant DNA)
• Genes to Make More Milk, Larger Eggs, More meat/less fat
3. Transgenic Plants: Contain Recombinant DNA
• Disease Resistant
• Larger Crop Yield
B. Cloning : Using One source of DNA to make Exact
copies
• Reproducing new individuals from one parent’s DNA
• Dolly
Dolly, the first Cloned Mammal
A donor cell is taken
from a sheep’s
udder.
Donor
Nucleus
These two cells are
fused using an
electric shock.
Fused Cell
Egg Cell
An egg cell is taken
from an adult female
sheep.
The nucleus of
the egg cell is
removed.
The fused cell
begins dividing
normally.
Embryo
Cloned Lamb
The embryo
develops
normally into a Foster
lamb—Dolly
Mother
The embryo is
placed in the
uterus of a foster
mother.
Flowchart
Cloning
A body cell is taken from a donor animal.
An egg cell is taken from a donor animal.
The nucleus is removed from the egg.
The body cell and egg are fused by electric shock.
The fused cell begins dividing, becoming an embryo.
The embryo is implanted into the uterus of a foster mother.
The embryo develops into a cloned animal.
Applications of biotechnology
I’m a very special pig!
Got any Questions?
2006-2007