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Genetic Engineering
AP Biology
Chapter 20
Terminology
• Genetic engineering – direct manipulation
of genetic material for practical purposes
• Biotechnology – use of living organisms or
their components to make products for us
• Recombinant DNA – combining pieces of
DNA from different organisms
• Gene cloning – making copies of DNA
Making recombinant DNA
• Plasmids (small circular pieces of DNA
in bacterial cells) are used to insert
pieces of foreign DNA
The DNA is cut using restriction
enzymes
What are restriction enzymes?
• Restriction enzymes come from
bacteria and recognize a particular
pattern of DNA, often 4, 6 or 8 base
pairs long, and then cut the DNA within
this recognized sequence.
• Bacteria use these enzymes to kill off
other competing bacteria by cutting up
their DNA.
How do they cut?
Bacterial Plasmid
chromosome
Gene of
Recombinant interest
DNA of
DNA (plasmid) chromosome
2 Plasmid put into
bacterial cell
Recombinant
bacterium
Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted
into plants up toxic waste attack therapy growth
Fig. 20-2a
Bacterial Plasmid
chromosome Gene of
Recombinant interest
DNA of
DNA (plasmid) 2 chromosome
2 Plasmid put into
bacterial cell
Recombinant
bacterium
Fig. 20-2b
Recombinant
bacterium
Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted
into plants up toxic waste attack therapy growth
Steps
1. Plasmid and human DNA are isolated.
2. Both DNAs are cut with the same
restriction enzyme.
3. “new” DNA is ligated into plasmid
4. Recombinant plasmids are inserted into
bacterial cells.
5. Plate bacteria on agar. Bacteria will
express new genes.
Nucleic Acid Hybridization
• Used to detect genes
• The DNA of the cell is denatured to
produce single stranded DNA.
• The radioactive probe will hybridize
(bond) with complementary bases if
present.
• Probes can be radioactive isotopes or
flourescent dyes.
The radioactive probe is
made by determining a
short segment of the
protein sequence, then
"back translating" to the
possible DNA sequences.
Short DNA sequences are
synthesized to match the
protein sequence. Then
these DNA oligomers
(known as "oligos") are
radiolabeled, and applied to
the blotted clones. They
should hybridize only to
clones containing
sequence encoding the
desired protein.
Expression of eukaryotic
genes in prokaryotes
• Use an expression vector with a prokaryotic
promoter upstream from the location of the gene.
• Create artificial genes without introns since bacteria
do not have the machinery for eliminating introns.
• YACS
What are YACS?
• Yeast artificial chromosomes that carry
foreign DNA.
A Thermocycler
Steps of PCR?
• Denature DNA (94-96 C)
• Anneal (base pair) primers (50 – 65 C)
• Extend primers (72 for polymerase to
work)
• Machines called thermocyclers do this.
http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
• In PCR, a heat-stable DNA polymerase is
used, most commonly Taq Polymerase
from the thermophilic microbe Thermus
aquaticus. Thomas Brock discovered T.
aquaticus from a hot spring at
Yellowstone National Park.
Why is PCR used prior to cloning
a gene in cells?
The task of later identifying
the clone carrying the gene
is simplified.
Applications of PCR
Gel Electrophoresis
Southern Blotting
DNA Fingerprinting
1. Isolate DNA
2. Cut DNA into fragments with restriction enzymes.
3. Electrophorese.
4. Blot onto nylon membrane.
5. Apply radioactive probes.
6. Wash to remove unbonded probes.
7. X-Ray.
Southern Blotting
Sanger Sequencing
• Used to sequence short segments of
DNA
• Fragments are incubated with
fluorescent dyes.
• When fragments hybridize with the
tagged nucleotide, the hybridization
stops.
• Fragments are electrophoresed and
analyzed.
Early DNA Sequencing
Analyzing Expression of Genes
• Northern Blotting, in situ hybridization –
using radioactive probes to look for mRNA
being produced
Fully differ-
Less differ- entiated
entiated cell (intestinal) cell
Donor Donor
nucleus Enucleated nucleus
trans- egg cell trans-
planted planted
Egg with donor nucleus
activated to begin
development
RESULTS
1 2
Egg cell
from ovary Nucleus
removed
Cultured 3 Cells fused
mammary cells 3
Nucleus from
mammary cell
4 Grown in
culture
Early embryo
5 Implanted
in uterus
of a third
sheep
Surrogate
mother
6 Embryonic
development Lamb (“Dolly”)
RESULTS genetically identical to
mammary cell donor
Why Dolly died young 6 yrs
• Dolly's telomeres were found to be
approximately 80% of the length they
should be for a sheep her age.
• Also there is the concern of damaged
DNA being carried into the clone
Cloned animals do not look exactly like
the transplanted nucleus due to
cytoplasmic affects.
CC
Rainbow
CC and her
Surrogate mom
• In most nuclear transplantation studies,
only a small percentage of cloned
embryos have developed normally to
birth
• Many epigenetic changes, such as
acetylation of histones or methylation of
DNA, must be reversed in the nucleus
from a donor animal in order for genes
to be expressed or repressed
appropriately for early stages of
development
Stem Cells
• Relatively unspecialized cells that
continue to reproduce themselves and
can be induced to form specialized
cells
DNA
T
Normal allele
SNP
C
Disease-causing
allele
Human Gene Therapy
Viral RNA
Bone
marrow
cell from
patient
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with RESULTS
the gene
of interest
Recombinant
Ti plasmid