1
Test
#1
-
Biotechnology
(Chapters
1
3
& 14)
Biotechnology
-
_______________________________________________________________________
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.
I.
Selective Breeding
–
Allowing only those organisms with desired traits (or without undesirable traits) to
produce offspring of the next generation.
A.
Inbreeding
-
____
________________________
__________________________________________
Examples:
____________
____________________________________________________________
Q:
What problem(s) can result from inbreeding?
___________
_______________________________
B.
Hybri
dization
-
__
_____________
_____________________________________________________
Examples:
_____
___________________________________________________________________
Q:
What i
s the benefit of hybridization
? _
________________________________________________
II. Genetic Enginee
ring
Gene
-
______________________________________________________________________________
Genome
-
_
__________________________________________________________________________
Genetic Engineering
(
recombinant
DNA technology)
–
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
humans
and bacteria).
Why i
s this possible
?
_______
___________
__________
____________________
II
I
.
Five
Stages of a Genetic Engineering Experiment
1.
DNA Extraction
–
DNA can be removed from most cells by opening the cells and separating the DNA from
the other cell parts.
2.
Cutti
ng DNA
–
Sequences of DNA are isolated
using
restriction enzymes
. T
hese enzymes attach
t
hemselves
to, and cut out, specific
sequences of DNA nucleotide
s (usually 4
-
6).
Restriction
enzymes
are
naturally
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
being cut
批
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
reattach.
†††
o
r could pair up with any other akA fragment or gene
that was
†††
cut by
the same restriction en穹me
.
2
Ligase
–
reforms the phosphodiester bonds.
100’s of diffe
rent restriction enzymes are known. Each e
ndonuclease recognizes only one
4
-
6 strand
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.
R
estriction enzyme
s are
used to cut or cleave the source DNA into fragments. The fragments are termed
RFLP’s
(Restriction Fragments Length Polymorphisms)
. Because the restriction enzyme
’s r
ecognition
sequence is lik
ely to
occur many times within the source
DNA, cutting will
produce many fragments of
different lengths. Dif
ferent
RFLP’s may be
made by using
different
restriction enzymes
that recognize
different DNA sequences.
3.
Sep
arating DNA
-
The RFLP’s can be separated (
based on their size
) by
electrophoresis.
Gel Electrophoresis (
DNA Fingerprinting
)
-
Sir Alec Jefferys from Great Britain
(
1980’s
)
Since a 3
-
billion
-
base sequence of the 4 DNA nucleotides can produce more varied
combi
nations than
there are humans,
each of us should have a unique DNA sequence.
The fingerprinting technique focuses on sequences of DNA that
_
_________________
____________
A.
The
Process
-
Gel electrophoresis is a procedure for separating a mixture of
molecules through a
stationary material (gel) in an electrical field.
1)
A gel is prepared which will act as a support for separation of the fragments of DNA. The gel is a
jello
-
like material, usually
agarose
, a substance derived from seaweed.
Holes (ca
lled
wells
) are created in the gel. These will serve as a reservoir to hold the DNA solution.
2)
DNA solutions (mixtures of different sizes of DNA fragments) are loaded in a well in the gel.
3)
The
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.
DNA ladders
are often electrophoresed with DNAs
.
They
are usually a
mixture of RFLPs
with known
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
other.
The DNA pieces, carrying
a negative charge because of the phosphate group,
______________
__
_
_____________________________________________
____________________________
The pieces migrate by
size, _______
_________
_____________________________________
We
l
l
3
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
DNA fingerprint
.
4.
Making Recombinant DNA
-
Once the DNA of interest is isolated,
it
is recombined with another organisms’ DNA
a)
To “recombine” or insert genes from one organism to
another organism, geneticists must first cut out the
desired gene using a restriction enzyme.
b)
With the same restriction enzyme, the geneticist cut
s
out a segment of DNA from a plasmid or a virus.
Plasmid’s
are
___________________________
_
_
__________________________________
__
____
_
c)
Because the two different sources of DNA (say, a human
and a bacteria) were cut w/the same
restricti
on
enzyme
, the “sticky” ends
will allow their DNA to recombine.
d)
Checking for
r
ecombinant cells
:
R
ecombinant molecules must be separated from molecules
consisting of just donor DNA or
plasmid DNA.
The experimenter designs the process so that the pl
asmid
contains two genes that each enable a cell to
grow in
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
t
hat are killed by one antibiotic but grow in the presence of the other contain
the p
lasmid with the foreign DNA.
5.
Cloning
a)
Plasmids (in bacteria) and viruses serve as vectors.
Vectors
are genetic vehicles that
______________
____________________________________________________
The host cell is usually a bacteria.
b)
As each “re
combined” cell reproduces, it forms cell clones that contain a copy of the inserted DNA
(gene).
IV
.
Genome Sequencing
–
Sequencing genomes is the process of locating all genes on all chromosomes of an
organism and
d
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.
In DNA
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,
4
replication of that strand stops producing a short fluores
cently color
-
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.
V.
Making Copies
-
PCR
–
Polymerase Chain Reaction is a man made proces
s of producing clones of DNA sequences
in a
machine.
After sequencing the nucleotides of the desired gene, primers are produced. Primers are single
-
stranded
sequences of DNA
nucleotides, which are used to copy both strands of target DNA (DNA in
tended
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
nucleotides together.
With the 2 strands separated, DNA polymerase
(enzyme) and nucleotides are added to the PCR machine
which is no
w
cooling down. As the temperature decreases,
DNA polymerase uses the primers as a starting point to
add free nuc
leotides
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
opying cycle.
Amplification (copying) continues until the desired numbers
of genes are made.
V
I
. Applications of Genetic Engineering
A. Transgenics
-
_____
___________
______________________________________________________
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,
that stra
nd is finished and shows
the color of the last base
added.
The color and length of each
fragment produced allows us to
“read” the sequence of the strand
of DNA.
5
1.
Transgenic Microo
rganisms
-
Used to produce
a variety of important substances useful for
health and industry. Ex.
-
insulin
2.
Transgenic Animals
-
Used to study genes and to improve the food
supply. Ex.
-
Mice with human genes that cause
their
immune syste
m to act like that of humans.
Scientists can use them to
study the effects
of
disease
on humans
3.
Transgenic Plants
-
The manipulation of
genes in farm animals
(Agrogenetics)
and plants to increase the amount
of food
produced and to reduce the chance of dying
due to infection and extremes in
temperature
and moisture.
V
I
I. Human Molecular Genetics
The exploration of human genes is now a major scientific
undertaking. Biologis
ts can now read, analyze and
even
change the
molecular code of genes.
A.
Reproductive Screening
-
_____________________
___________________
____________________
Ex.
-
Amniocentesis
-
____
_________________________
_______________________________
From this fluid, t
he fetuses DNA can be acquired and
analyzed.
B.
DNA Fingerprinting
-
The identification
of organisms using sequences of DNA that varies widely between
organisms.
Q:
What are some uses of this process?
_________
________________________________________
_______________________________________________________________________________
6
Example Problem:
Is Jack the father of Payle? _____________
How do you know?
____
_____
____________
_____
___
_____
______
________________________
Example Problem:
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,
and
D
. 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.
________________________
1
.
Which Plant Specie(s) has the
smallest fragment of
DNA? ____
_
_
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? _________
C.
The Human Genome Project
-
an international effort to map and sequence the human genome. This project
began in 1990 und
er
the leadership of James Watson at a co
st of
$
3
,000,000.
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
s)?
________________________________________
_______
_________________________________________________________________________
____
D.
Gene Therapy
–
A therapy used to cure a diseased individual who does not possess a copy of a necessary
gene.
(
A g
ene that
makes a protein
,
such as
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
+
–
+
←
Well
Q: What
determines
how far a
fragment will move in the gel?
=
RFLP
7
Normal hemoglobin gene
Chromosomes
Genetically engineered virus
Nucleus
Bone
marrow
c. direct insertion into affected cells
V
III.
Stem Cells
–
Unspecialized cells th
at can produce daughter cells that
are specialized (have specific
functions). Stem cells are classified by their
plasticity
, or ability to
become other, specific
cells.
Adult humans are made up of over 200 different specialized cells (skin, liver, heart,
etc)
Stem cell categories include:
1.
Totipotent
–
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.
2.
Pluripotent
–
P
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
tent cells.
3.
Multipotent
-
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.
4.
Adult
–
An adult
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
problems.
Bone marrow cell
8
IX
. Bioethics
–
A. Bioethics includes people’s response to the ever growing understand
ing and use of gene technologies and cell
reproduction.
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
al issues.
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
Genetic Engineeri
ng Application
Pros
Cons
Mammal Cloning
(sheep, pigs, humans)
* Replacement organs (i.e.
–
new
heart or liver for those suffering
from liver or heart failureF without
rejection
DNA Fingerprinting
* False positives
* Human error
Human Genome Proje
ct
Reproductive Screening
(amniocentesis)
Agrogenetics
Gene Therapy
Stem Cell Research
9
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?
The Viewpoints
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.
DNA Inform
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
databases.
You Decide
1.
What are the major issues regarding DNA databases?
2.
Are there any circumstances in which an employer might be justified in demanding DNA samples from its
emplo
yees? Why might an employee wish to withhold such samples?
3.
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?
4.
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?
10
Test #
1
Format: Bi
otech
Test Date: _________________
Multiple Choice from the notes
:
Plasmid: _______________________________________
__________
____________________________________
RFLP: _______________________________________
____________
_________
__________________________
Ligase: ____
_________
________________________________________________________________________
Restriction enzymes: __
_________
________________________________________________________________
Genetic en
gineering: ____________________
_________
______________________________________________
Recombinant DNA Technology: ____________________________________________________________________
PCR: __
______________________________________
_________
____________
_________________________
_
Gene: ______________________________________________________________________________________
Genome: ___________
_________________________________
_
_____________________________
__________
DNA Fingerprinting: ________________
______________________
_________
____________________________
Gene sequencing: __________________________________________________________
_________
___________
Cloning: _________
_________
___________________________________________________________________
Gene therapy: _____________
_________
__________________________________________________________
Transgenics: _______________________
_________
_________________________________________________
Vector: ________________________________________________
_____
____
____________________________
Written Questions
–
Why is it possible to transfer DNA between completely unrelated organisms?
What is the function of restriction enzymes in bacteria?
Describe
how restriction enzymes are used in a genetic engineer
ing experiment.
Describe
the process of Gel electrophoresis.
Describe
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
the
n reimplanted, would the change
be passed on to the patient’s children? Explain.
How are transgenic organisms made?
How have they helped humans?
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