Test #1B- Mendelian Genetics, Genetic Engineering and the Human ...

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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


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?