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Biotechnology and

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?

STICKY ENDS BLUNT ENDS


ACT GAA TTC CGG AAT GAA TTC
TGA CTT AAG GCC TTA CTT AAG

Where would the enzyme EcoRI cut?


ACT GAA TTC CGG AAT GAA TTC
TGA CTT AAG GCC TTA CTT AAG

There would be three pieces:


one 4 bases,
one 12 bases, and
one 5 bases.
Making recombinant DNA in
plasmids
The collection of thousands of
clones of bacteria containing
recombinant plasmids is called a
genomic library.
Genes can be
cloned into
vectors such
as plasmids
Fig. 20-2
Cell containing gene
Bacterium of interest
1 Gene inserted into
plasmid

Bacterial Plasmid
chromosome
Gene of
Recombinant interest
DNA of
DNA (plasmid) chromosome
2 Plasmid put into
bacterial cell

Recombinant
bacterium

3 Host cell grown in culture


to form a clone of cells
containing the “cloned”
gene of interest

Gene of Protein expressed


Interest by gene of interest
Copies of gene Protein harvested

4 Basic research and


Basic various applications Basic
research research
on gene on protein

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

Cell containing gene


Bacterium
of interest
1 Gene inserted into
plasmid

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

3 Host cell grown in culture


to form a clone of cells
containing the “cloned”
gene of interest

Gene of Protein expressed


Interest by gene of interest

Copies of gene Protein harvested

4 Basic research and


Basic various applications Basic
research research
on gene on protein

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.

• Yeast cells have plasmids that can act


as vectors.
Electroporation
• injecting DNA into eukaryotic cells
PCR Polymerase Chain Reaction
• Used to amplify DNA
• Discovered by Kary Mullis (GT grad)

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

PCR has replaced cloning for many


purposes, particularly the sequencing of
DNA.
• It is faster and requires no vectors, which
can mutate as they reproduce.
• It can be used forensically, to amplify tiny
amounts of DNA from criminal evidence; or
clinically, to detect DNA sequences linked to
inherited disorders.
What is gel electrophoresis?
• A technique to separate DNA based on
the movement of DNA fragments from
neg to pos (DNA is neg).

• Smaller fragments travel farther.

• Samples are placed in gels.

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

• RT-PCR – Reverse transcriptase-


polylmerase chain reaction – makes cDNA
from mRNAs and then PCRs the DNA for
electrophoresis
• Micro – arrays - Isolate mRNA from
cells, make cDNA using reverse
transcriptase, then uses cDNA to
explore collections of genomic DNA
• Microarrays are useful in discerning
gene expression in different tissues
AND at different stages of
development.
• Different brightness and
colors signify rates of
expression.
Determining Gene Function
• In vitro mutagenesis – changes made
to cloned gene, gene returned to cell
and it “knocks out” the normal gene.
Then look for abnormalities.

• RNA interference (RNAi) – uses double


RNA to block translation of mRNA.
Cloning Organisms
• Organismal cloning – producing
genetically identical individuals from a
single somatic cell of a multicellular
organism
In plants
• Steward demonstrated
genomic equivalence in
plants by growing
carrot plants from
differentiated root cells.

• Most plant cells remain


totipotent, retaining the
ability to give rise to a
complete new
organisms.
In Animals
• Briggs and all transplanted nuclei from
embryonic frog cells into enucleated
egg cells and produced cloned frogs
• Nuclear transplantation – name of
process
• Whether normal development occurred
depended on developmental age of the
transplanted nucleus.
Fig. 20-17
EXPERIMENT Frog embryo Frog egg cell Frog tadpole
UV

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

Most develop Most stop developing


into tadpoles before tadpole stage
Nuclear Transplantation
And then Dolly came along in
1997
Fig. 20-18
TECHNIQUE

Mammary Egg cell


cell donor donor

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

• Embryonic cells are more totipotent


than adult stem cells
• Therapeutic cloning – using stem cells to
replace organs and tissues

• Reproductive cloning – using stem cells to


reproduce new organisms

• Both raise ethical


debates
Benefits of DNA technology
• Medical Applications

• identification of human genes in which


mutation plays a role in genetic
diseases
• Single nucleotide polymorphisms
(SNPs) are useful genetic markers
• These are single base-pair sites that
vary in a population
• When a restriction enzyme is added,
SNPs result in DNA fragments with
different lengths, or restriction fragment
length polymorphisms (RFLP)
Fig. 20-21

DNA
T
Normal allele
SNP

C
Disease-causing
allele
Human Gene Therapy

• Gene therapy is the alteration of an


afflicted individual’s genes
• Vectors, such as viruses, are used for
delivery of genes into specific types of
cells, for example bone marrow
• It may be difficult to target cells.
• Gene therapy raises ethical questions,
such as whether human germ-line cells
should be treated to correct the defect
in future generations
Fig. 20-22
Cloned
gene
1 Insert RNA version of normal allele
into retrovirus.

Viral RNA

2 Let retrovirus infect bone marrow cells


Retrovirus that have been removed from the
capsid patient and cultured.

3 Viral DNA carrying the normal


allele inserts into chromosome.

Bone
marrow
cell from
patient

4 Inject engineered Bone


cells into patient. marrow
Pharmaceutical Products
• Advances in DNA technology and
genetic research are important to the
development of new drugs to treat
diseases
• In particular “pharm” animals and
plants can be used to produce
certain products
Fig. 20-23
Forensic Evidence and Genetic
Profiles
• An individual’s unique DNA sequence, or
genetic profile, can be obtained by analysis of
tissue or body fluids

• Even more sensitive is the use of genetic


markers called short tandem repeats (STRs),
which are variations in the number of repeats
of specific DNA sequences
Fig. 20-24
(a) This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.

Source of STR STR STR


sample marker 1 marker 2 marker 3

Semen on victim 17, 19 13, 16 12, 12

Earl Washington 16, 18 14, 15 11, 12

Kenneth Tinsley 17, 19 13, 16 12, 12

(b) These and other STR data exonerated Washington and


led Tinsley to plead guilty to the murder.
Environmental Cleanup
• Some modified microorganisms can
be used to extract minerals from the
environment or degrade potentially
toxic waste materials
• Biofuels make use of crops such as
corn, soybeans, and cassava to
replace fossil fuels
Genetic Engineering in Plants
• Agricultural scientists have endowed a
number of crop plants with genes for
desirable traits
• The Ti plasmid is the most commonly
used vector for introducing new genes
into plant cells
• Most public concern about possible hazards
centers on genetically modified (GM)
organisms used as food
• Some are concerned about the creation of
“super weeds” from the transfer of genes
from GM crops to their wild relatives
Fig. 20-25
TECHNIQUE

Agrobacterium tumefaciens

Ti
plasmid

Site where
restriction
enzyme cuts
T DNA
DNA with RESULTS
the gene
of interest

Recombinant
Ti plasmid

Plant with new trait