 |
 |
 |
 |
 |
 |
 |
 |
 |
|
K-12
Programs
|
||
Genetic Counseling
Background It is probable that at some time in the
future, you will have a genetic analysis of your genome
conducted. Most likely,
this will be to screen for a disease.
Genetic tests might determine if you have a disease or the
potential to develop a disease, or if you carry a disease trait you
might pass on to your offspring.
In addition, the screen might provide information about which
designer drug would be most
effective for you. You
will provide a blood sample and DNA from the white
blood cells in the blood sample will be collected for
analysis. A small
amount of DNA will be subjected to Polymerase Chain Reaction (PCR)
amplification with primers specific for the disease
allele.
Alleles are different versions of a gene, like the same piece
of clothing in different sizes or colors.
They come in a variety of forms, some with differences as
small as a single base change, others with deletions (bases
missing), insertions (extra bases) and inversions (sections of
flipped DNA). The size
of the mutation does not necessarily determine the severity of the
effect. For example, sickle cell anemia is caused by a single point
mutation which creates a mutant form of hemoglobin that
cannot fold or function properly. In some cases, a gene will naturally carry a repeat region.
Alleles of these genes often carry varying numbers of
repeats. The repeated
areas are referred to as variable nuclear tandem repeats (VNTRs) and
can be used to identify individuals.
Some diseases have been linked to the number of repeats.
For example, in Huntington’s
disease the severity and time of onset of the disease has been
linked to the number of repeats of a certain short sequence.
Definitions: Genome – complete collection of an
organism’s DNA Designer drugs – drugs which have been
specifically designed to work for a specific genome.
These drugs are not currently available, but are predicted to
revolutionize the effectiveness of medications.
White blood cells – immune cells.
Blood is composed of red and white blood cells, platelets and
plasma. The white blood cells are the only component of blood with
DNA. Allele – variant of a gene Gene – a section of DNA encoding a protein
or RNA Point mutation – a single nucleotide change
in a gene Huntington’s – a fatal neurodegenerative
disease Disclaimer The following scenario is made up.
There is no gene for “coolness”.
Social popularity is a complex, unstable phenomenon based on
multiple genetic and environmental factors with mixed selective
results. Which means
that what’s cool in Wisconsin today, may not be tomorrow, and may
never have been cool in Tokyo.
However, all the molecular biology techniques are real, and
the overall genetic analysis procedure follows the same steps as a
real analysis would. The Scenario Back to the scenario… Unbeknownst to the candidates, you begin
collecting their DNA samples. Candidate
A’s younger brother is bribed into bringing you his sibling’s
toothbrush. Candidate B
has a couple hairs tugged out during wrestling in PE by your
assistant. You snag
candidate C’s buccal (cheek cell) swab in biology class.
1) Is it ethical to collect and test DNA samples without the subject’s permission or knowledge? Are there circumstances under which you might change your mind? For example, would DNA sampling without permission be acceptable to solve a serial murder case? What about a national health study investigating possible treatments for diabetes?
You take this motley collection of illicitly
obtained body parts to lab and isolate DNA from each sample. 2)
What cell type will yield DNA from each sample?
Which sample will yield the most DNA?
Why?
As control samples, you collect DNA from
people you know are very cool (the prom king and queen, hot local DJ
and a rapper, or the head of the debate society), and people who
rank low on the cool scale (your younger sibling or Great Aunt Ida).
3) Why is it
important to use controls? What
information do the controls provide?
What is the difference between a positive and a negative
control? In this scenario,
which samples provide the positive control?
And the negative control?
PCR: Since the quantity of DNA obtained is so
small, each sample must be amplified before it can be analyzed
further. The samples
will be amplified using the Polymerase Chain Reaction or PCR.
PCR is a straightforward process which can generate a large
quantity of DNA from a small sample of DNA or RNA in a short time.
The sample DNA is denatured (the two strands are separated),
by heat. After
denaturing the template (original) DNA, the sample is cooled and the
primer is added. The
basic concept is that a small piece of DNA called a primer is
designed to anneal to a certain section of the DNA sample.
For example, since you are investigating the cool gene, you
would design a primer which anneals to a section of the cool gene.
A second primer which anneals to the opposite strand
downstream of the first primer is also designed.
DNA polymerases are a class of proteins which
build a strand of DNA from nucleotides (G,A,T,C) by pairing them to
the template DNA. In
PCR, this is called extension or elongation.
A DNA polymerase in the reaction mixture will generate a new
strand of DNA starting from each primer.
Then the new DNA strands and the original DNA templates are
denatured by heating the reaction.
This is where the story would end if it were
not for the discovery of a thermostable DNA polymerase.
Bacteria called thermophiles (heat lovers) live in extremely
hot environments, such as thermal vents and hot springs.
Most organisms cannot live in extreme heat because their
proteins denature (unfold) and cease to function.
Naturally, the proteins in a thermophile are resistant to
denaturation by heat. The DNA polymerase from the thermophile T. aquaticus
is used to build new DNA strands in PCR because the denaturation
step used to separate the DNA strands does not affect the polymerase.
Therefore, the steps can be repeated many times, each time
with more template DNA available for primer annealing and extension
by polymerase. The
quantity of DNA increases exponentially generating usable quantities
of high quality DNA in a relatively short time.
3)
Could PCR be conducted with DNA polymerase from another
organism? If so, which
organism(s) would be good candidates? If not, why not? 4)
What does PCR allow us to do that we previously could not?
Why is this important? Electrophoresis: The DNA generated by your PCR experiments
will contain the amplified fragment of the cool gene.
There are several alleles of this gene which vary in the
number of repeats of a certain area.
(Go back and read that again until you are VERY clear about
it. If you are having
trouble seeing exactly what this means, ask for help.)
In order to determine which allele each person has, you must
be able to see the DNA and determine the size of each amplified
fragment. To do this,
you will use electrophoresis. DNA
has an overall negative charge from the phosphate groups; therefore
DNA in an electrical field will move toward the positive pole of an
electrical field. If the DNA is forced to move through a
molecular sieve, which works exactly like your kitchen sieve only
much smaller; small fragments move through the sieve more quickly
than large fragments. Fragments
of the same size will move at the same rate resulting in “bands”
of DNA. The molecular
sieve is provided by an agarose gel.
Agarose is a large sugar polymer.
More agarose makes a tighter sieve while less makes a looser
sieve. Agarose is used
as a thickener in many foods, and you will probably recognize the
similarity to jello. Do
not attempt to eat your gel. It
will not taste good and contains toxic elements.
To generate the electrical field to pull the
DNA through the gel, the gel is submerged in a buffer which will
conduct an electrical current. To allow you to see the DNA, ethidium bromide, which is a dye
that sticks to DNA is added to the gel.
Ethidium bromide is a carcinogen (cancer causing agent)
precisely because it sticks to DNA.
Do not handle the gel or buffer without gloves.
Ethidium bromide glows red when illuminated by UV light
allowing you to see (by inference) where the DNA bands are in the
gel. Follow the
protocol on pages 2 and 3 of your procedure for electrophoresis. 5)
Why can’t you see the DNA itself? 6)
Which bands move the farthest down the gel, bands containing
large fragments or bands of small fragments?
Why? Analysis: Use the results to determine which person is
the coolest. Compare
the control DNA samples to the subjects’ samples.
Remember that “coolness” is directly related to the
number of repeats in the “cool” gene, so more repeats means a
cooler person. 7)
Will the bands from the cool people or the not-too-cool
people run farthest down the gel? 8)
If the repeat is approximately 50bp, how many copies of the
repeat does each person in your assay have?
|
||
| FOR MORE INFORMATION | ||
|
Contact: Barbara Bielec, 608-273-9737 |
||
© 2002, BTCI. All rights reserved. If you encounter technical problems with this site, please e-mail the webmaster. |
||
 |
 |