For thousands of years, humans have used selective breeding to pass desired traits on to the next generation in
our domestic plants and animals. Selective breeding is limited to the traits or genetic variation that naturally
exists in the species (you can’t select for dogs with wings since they don’t have genes for wings!) Biotechnology and
Genetic engineering now
allow for genes to be moved from one organism to another.
Allowing only those organisms with desired traits (or without undesirable traits) to
produce offspring of the next generation.
What problem(s) can result from inbreeding?
s the benefit of hybridization
II. Genetic Enginee
The transferring of DNA/gen
es from organism to
another. Also called
gene splicing. Genetic engineering
can take place within a species
(switching genes between humans) or
between species (switching genes between
s this possible
Stages of a Genetic Engineering Experiment
DNA can be removed from most cells by opening the cells and separating the DNA from
the other cell parts.
Sequences of DNA are isolated
hese enzymes attach
to, and cut out, specific
sequences of DNA nucleotide
s (usually 4
occurring in bacteria.
What is the role/function of
restriction enzymes in bacteria?
What is the role of restriction enzymes for genetic engineering?
DNA fragments or genes are said
to be “sticky” after
restriction en穹mes. qhey become stic歹 because instead of
cutting the 4
6 nucleotide sequence in the centerI the cut is
made to one side leaving twoI single complementary strands.
qhe cut ends Ebecause
they are complementaryF can
r could pair up with any other akA fragment or gene
the same restriction en穹me
reforms the phosphodiester bonds.
100’s of diffe
rent restriction enzymes are known. Each e
ndonuclease recognizes only one
nucleotide sequence and always cuts it at the same place. By trial and error, geneticists usually find a
sequence of DNA nucleotides on either side of the desired gene,
and not within the gene, that can be cut
by restriction enzymes.
used to cut or cleave the source DNA into fragments. The fragments are termed
(Restriction Fragments Length Polymorphisms)
. Because the restriction enzyme
sequence is lik
occur many times within the source
DNA, cutting will
produce many fragments of
different lengths. Dif
RFLP’s may be
made by using
different DNA sequences.
The RFLP’s can be separated (
based on their size
Gel Electrophoresis (
Sir Alec Jefferys from Great Britain
Since a 3
base sequence of the 4 DNA nucleotides can produce more varied
there are humans,
each of us should have a unique DNA sequence.
The fingerprinting technique focuses on sequences of DNA that
Gel electrophoresis is a procedure for separating a mixture of
molecules through a
stationary material (gel) in an electrical field.
A gel is prepared which will act as a support for separation of the fragments of DNA. The gel is a
like material, usually
, a substance derived from seaweed.
) are created in the gel. These will serve as a reservoir to hold the DNA solution.
DNA solutions (mixtures of different sizes of DNA fragments) are loaded in a well in the gel.
gel matrix acts as a sieve for DNA molecules. Large molecu
les have difficulty getting through
the holes in the matrix. Small molecules move easily through the holes
Because of this, large
fragments will lag behind small fragments as DNAs migrate through the gel.
are often electrophoresed with DNAs
are usually a
mixture of RFLPs
lengths and are
used to estimate the sizes of DNA fragments in your DNA sample
A positive electrode is applied to one end of the gel and a negative electrode to the
The DNA pieces, carrying
a negative charge because of the phosphate group,
The pieces migrate by
The pattern th
at forms from the different sized fragments, with the shorter pieces close
r to the positive
pole and the
longer fragments further away, is a
Making Recombinant DNA
Once the DNA of interest is isolated,
is recombined with another organisms’ DNA
To “recombine” or insert genes from one organism to
another organism, geneticists must first cut out the
desired gene using a restriction enzyme.
With the same restriction enzyme, the geneticist cut
out a segment of DNA from a plasmid or a virus.
Because the two different sources of DNA (say, a human
and a bacteria) were cut w/the same
, the “sticky” ends
will allow their DNA to recombine.
ecombinant molecules must be separated from molecules
consisting of just donor DNA or
The experimenter designs the process so that the pl
contains two genes that each enable a cell to
the presence of a different antibiotic drug.
When the piece of DNA to be cloned (mass produced) inserts into the plasmid,
The researcher can then tell which cells have taken up the plasmid by exposing the cells to each
antibiotic. Only bacteria
hat are killed by one antibiotic but grow in the presence of the other contain
lasmid with the foreign DNA.
Plasmids (in bacteria) and viruses serve as vectors.
are genetic vehicles that
The host cell is usually a bacteria.
As each “re
combined” cell reproduces, it forms cell clones that contain a copy of the inserted DNA
Sequencing genomes is the process of locating all genes on all chromosomes of an
eciphering each genes nucleotide or base sequence.
The number of completed genomes is approaching 200! The list includes members of all 6 kingdoms
(Archaebacteria, Eubacteria, Protists, Fungi, Plants, & Animals), viruses, and cellular organelles.
sequencing, a complementary DNA strand is made using a small portion of fluorescently labeled
(glowing colors) nucleotides. Each time a labeled nucleotide is added in place of a normal nucleotide,
replication of that strand stops producing a short fluores
coded DNA fragment. When the
mixture of fragments is separated on a gel, the DNA sequence can be read directly from the gel based on
those fluorescent colors.
Polymerase Chain Reaction is a man made proces
s of producing clones of DNA sequences
After sequencing the nucleotides of the desired gene, primers are produced. Primers are single
sequences of DNA
nucleotides, which are used to copy both strands of target DNA (DNA in
to be copied).
A solution of target DNA and primer are added to a PCR machine and heated to a temperature warm enough
to break the
hydrogen bonds holding the two strands of DNA together, but not too hot to break
the other bonds holding the
With the 2 strands separated, DNA polymerase
(enzyme) and nucleotides are added to the PCR machine
which is no
cooling down. As the temperature decreases,
DNA polymerase uses the primers as a starting point to
add free nuc
down the strand of DNA. When it
is complete, what used to be the primer is now lengthened
into a complementary copy of the entire gene.
Because both strands are copied in this process, there are
now 2 copies of the original gene after one c
Amplification (copying) continues until the desired numbers
of genes are made.
. Applications of Genetic Engineering
A different color fluorescent dye
is used for each base (A,C,G,T).
When a dyed base is added to a
strand instead of a normal one,
nd is finished and shows
the color of the last base
The color and length of each
fragment produced allows us to
“read” the sequence of the strand
Used to produce
a variety of important substances useful for
health and industry. Ex.
Used to study genes and to improve the food
Mice with human genes that cause
m to act like that of humans.
Scientists can use them to
study the effects
The manipulation of
genes in farm animals
and plants to increase the amount
produced and to reduce the chance of dying
due to infection and extremes in
I. Human Molecular Genetics
The exploration of human genes is now a major scientific
ts can now read, analyze and
molecular code of genes.
From this fluid, t
he fetuses DNA can be acquired and
of organisms using sequences of DNA that varies widely between
What are some uses of this process?
Is Jack the father of Payle? _____________
How do you know?
Scientists found members of a plant species they did not recognize. They wanted to determine if the unknown
species was related to one or more of four known species,
A, B, C,
. The relationship between sp
ecies can be
determined most accurately by comparing the results of gel electrophoresis of the DNA from different species.
The chart below represents the results of gel electrophoresis of the DNA from the unknown plant species and the
four known species.
Which Plant Specie(s) has the
smallest fragment of
2. Which Plant Specie(s) has the largest
fragment of DNA? ___________
3. Which Plant Specie(s) is most closely related
to the unknown plant? _________
4. Which Plant Specie(s) is least closely related to the unknown plant? _________
The Human Genome Project
an international effort to map and sequence the human genome. This project
began in 1990 und
the leadership of James Watson at a co
It was finished in 2002
Q: What are some uses for the information gained from the Human Genome Project (and from
sequencing other organisms’ genome
A therapy used to cure a diseased individual who does not possess a copy of a necessary
makes a protein
insulin, to control blood sugar.)
1. A normal gene is cut out using restriction enzymes and copied by PCR (Polymerase Chain Reaction).
2. The copies are introduced into the diseased individual.
3. Methods for intr
oducing the gene include
a. using non
harmful viruses (vector) to deliver gene to a cell’s DNA
b. intravenous (IV) injections into the bloodstream
how far a
fragment will move in the gel?
Normal hemoglobin gene
Genetically engineered virus
c. direct insertion into affected cells
Unspecialized cells th
at can produce daughter cells that
are specialized (have specific
functions). Stem cells are classified by their
, or ability to
become other, specific
Adult humans are made up of over 200 different specialized cells (skin, liver, heart,
Stem cell categories include:
The most plastic or versatile stem cell. When a sperm cell and egg cell unite during fertilization,
the result is a one
celled zygote. The zygote is totipotent because it can give rise to any cell t
ype, including an
entire organism. The zygote will eventually become every cell of an organism including other stem cells. The
first few cell divisions make more totipotent cells, after 4 days the divisions produce pluripotent cells.
luripotent cells, like totipotent cells can give rise to any type of cell. Unlike totipotent cells,
pluripotent cells cannot create an entire organism. Also on day 4 of development, the embryo forms into 2
layers. The outer layer becomes the placenta (co
nnect embryo/fetus to mother), and the inner layer will
become the organism. Though the inner cells can become any type of human cell, it cannot without the outer
layer of cells, thus pluripotent and not totipotent. Pluripotent cells give rise to multipo
These cells are less plastic. They will become one of a few types of cells within a particular
tissue. For example, multipotent blood cells can become red blood cells, white blood cells, or platelets.
stem cell is a multipotent stem cell in adult humans that is used to replace cells that have been
damaged, infected, or died. Adult stem cells are unspecialized cells in specialized tissue.
Stem Cell Research
Therapeutic cloning produces stem cells whic
h can develop into different types of body cells, making them ideal for
research into treatment of disease. But this technology involves the creation and destruction of embryos, which is
ethically controversial. The stem cells created also run the risk of
being rejected by the body. The new
technology, nuclear reprogramming, creates stem
like cells from the patient's own cells, avoiding both these
Bone marrow cell
A. Bioethics includes people’s response to the ever growing understand
ing and use of gene technologies and cell
B. Because of the wide variety of cultures and societies, there is a wide variety in personal ethics, causing many
bioethical beliefs and concerns. This can lead to spirited debate about biologic
C. Examples of bioethical issues: stem cell research, genetically engineered foods
Q: Who determines which technologies are carried out in a society?
Pros and Cons of Genetic Engineering
(sheep, pigs, humans)
* Replacement organs (i.e.
heart or liver for those suffering
from liver or heart failureF without
* False positives
* Human error
Human Genome Proje
Stem Cell Research
Who Controls Your DNA?
April 16. Cpl. John C. Mayfield and Cpl. Joseph Vlacovsky were found guilty of disobeying a lawful order. The U.S.
Department of D
efense requires DNA samples for a database that could be used to identify soldiers’ remains. The
two Marines refused.
At their court martial, the two Marines argued that DNA samples could be examined for genes related to disease
or even behavior and, there
fore, the database was an invasion of privacy. As a result of the concerns raised by this
case, the U.S. Department of Defense has changed its policies. It now destroys DNA samples upon request when an
individual leaves military service. Do people have a r
ight to control their own DNA samples?
DNA Information Is Not Private
As the court recognized, the U.S. Department of Defense had good reasons for requiring that DNA samples be
taken and stored. Furthermore, DNA sequences are no more priv
ate and personal than fingerprints or photographs,
which are taken by private and government agencies all the time. An employer has a right to take and keep such
information. Individuals should have no reason to fear the abuse of such databases.
ation Is Private and Personal
The use of DNA for personal identification by the military may be justified. An individual’s genetic information,
however, is a private matter. A recent study at Harvard and Stanford universities turned up more than 200 cases
of discrimination because of genes individuals carried or were suspected of carrying. Employers with DNA
information might use it to discriminate against workers who carry genes they suspect might cause medical or
behavioral problems. Individuals must hav
e the right to control their own DNA and to withhold samples from such
What are the major issues regarding DNA databases?
Are there any circumstances in which an employer might be justified in demanding DNA samples from its
yees? Why might an employee wish to withhold such samples?
Should the control of DNA databases be a matter of law, or should it be a matter to be negotiated
between people, their employers, and insurance companies?
Suppose you were a doctor working
as a consultant to a health insurance company. The insurance company is
trying to decide whether to test adults for cystic fibrosis alleles before agreeing to insure their families.
What advice would you give to the company about this?
Test Date: _________________
Multiple Choice from the notes
Restriction enzymes: __
Recombinant DNA Technology: ____________________________________________________________________
DNA Fingerprinting: ________________
Gene sequencing: __________________________________________________________
Gene therapy: _____________
Why is it possible to transfer DNA between completely unrelated organisms?
What is the function of restriction enzymes in bacteria?
how restriction enzymes are used in a genetic engineer
the process of Gel electrophoresis.
multiple ways that DNA fingerprinting can be used.
Describe what happens during a polymerase chain reaction.
If a human patient’s bone marrow were removed, genetically altered, and
n reimplanted, would the change
be passed on to the patient’s children? Explain.
How are transgenic organisms made?
How have they helped humans?