Download Lab 3 In Search of the Sickle Cell GeneSp08

Dangerous Ideas and Forbidden Knowledge, Spring 2008
Lab 3
Part 1: In Search of the
Sickle Cell Gene
OBJECTIVES:
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Become more familiar with the structure and function of red blood cells
Review and practice our microscopy skills
Improve our understanding of the general structure and function of DNA
Learn how to use micropipettes
Understand and perform DNA electrophoresis
Understand and perform a Southern Blot
Improve our understanding of Mendalian Genetics
Think about natural selection acting on human populations
Understand how a gene specifies a particular protein (transcription and translation)
General Background:
DNA contains the directions for making proteins, carefully encoded in a series of bases
or “letters”. Mutations can alter these directions, occasionally having dramatic impacts on the
structure of the proteins encoded by our DNA. This is the basis for most inherited diseases. And
thanks to the work of the Augustine monk, Gregor Mendel, we can now predict how many such
mutations will be inherited. Over the next several weeks, we will be using the example of Sickle
Cell Disease to explore Mendalian genetics, DNA technology, human evolution, and how the
directions encoded in a molecule of DNA are translated into proteins.
Sickle Cell Disease results from inheriting two copies
of a mutated hemoglobin gene, one from your mother, and one
from your father. Hemoglobin is a critical protein as it serves
as the carrier of oxygen. A single human red blood cell
contains up to 250 million molecules of the hemoglobin
protein, each carrying up to four molecules of oxygen (O2)!
The sickle cell mutation results from just one “letter” in the
DNA chain being substituted for another. This single change
dramatically affects the structure of the protein. Mutated
hemoglobin proteins clump together in their unoxygenated
state, distorting both the structure and function of the red
blood cells. (The image at right shows both normal plump,
symmetrical red blood cells and a ‘sickled’ red blood cell.)
Children inheriting two copies of the sickle cell gene suffer a variety of effects including
increased risk of infections, slow growth rates, acute pain episodes, eye damage, swelling in the
hands and feet, poor circulation, and increased risk of stroke. Interestingly, individuals with one
normal copy of this gene and one mutated copy (heterozygotes) usually have no symptoms and
normal lifespans.
The Sickle Cell story gets even stranger when we survey the human population, looking
for this particular, mutated gene. The Sickle Cell gene is carried by about 8% of African
Americans, and virtually absent in most other ethnic groups. This is puzzling as evolutionary
biologists typically expect harmful mutations to be “selected out” of a population. Why is the
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Dangerous Ideas and Forbidden Knowledge, Spring 2008
frequency of this gene so high in African Americans? And why is it rarely seen in most other
groups?
The answer to these questions may lie, in part, in the tiny Anopheles mosquito. This
mosquito can transmit malaria, a devastating human parasite. The U.S. Center for Disease
Control (CDC) estimates that, globally, 700,000-2.7 million people a year die from malaria, and
that 75% of these deaths are in African children. When we map the incidence of malaria, there is
a striking correlation with the frequency of the sickle cell hemoglobin gene. In areas of the world
where malaria is endemic, this mutant version of the gene (allele) is much more common.
Further studies have confirmed that individuals who carry one normal copy of the hemoglobin
gene, and one mutant copy, are more resistant to the malarial parasite, and more likely to survive
an infection. Thus this simple mutation may have provided a powerful selective advantage in
malarial prone areas.
Overview of Procedures:
In lab today, we will be working with DNA from two hypothetical parents. These
parents would like to test their DNA, and their child’s DNA, for the Sickle Cell gene. There are a
variety of ways such a test could be conducted, including through the use of a Southern Blot.
To conduct a Southern Blot, researchers would first obtain DNA samples from the
parents and child. These samples could come from a blood draw, cheek cells, or even hair
follicles. The DNA extraction is easy, but finding the gene you’re looking for is much more
problematic. We currently estimate that humans have at least 35,000 genes scattered across their
chromosomes. How will we find the sickle cell gene?
To find this specific gene, the DNA will first need to be cut into many small pieces.
Molecular biologist using naturally occurring enzymes, called restriction enzymes, to do this
cutting. Conveniently, each restriction enzyme can cut DNA only at very specific sequences. As
all individuals have slightly different DNA sequences, their DNA will be cut at slightly different
places. In particular, one of the enzymes will cut the normal hemoglobin gene, but not the
mutated form! The DNA you will be given today, has already been cut. Thus we are starting
with a collection of differently sized pieces from each individual. Our task will be to sort through
these fragments and find the sickle cell gene!
We’ll sort these fragments by using a
technique called DNA electrophoresis. In DNA
electrophoresis, a collection of DNA molecules is
placed in wells at one end of a gel. An electrical
current is then conducted across the gel. As DNA
is a negatively charged molecule, it will be drawn
towards the positive pole. (Opposites attract!)
Thus our DNA will move through the gel, and
smaller fragments will travel faster, and therefore
farther, than larger fragments. This allows us to
sort the DNA fragments from each individual based
on their size. Note that we still have thousands of
pieces of DNA, and don’t know which on is our
sickle cell gene. But at least we have them
organized!
Our next step, finding the gene, will involve transferring this nicely sorted collection of
DNA fragments to a more durable membrane. With our DNA fragments affixed to this
membrane, we can then start searching for our gene of interest. This process of transferring DNA
to a membrane and probing it for a particular gene is known as a Southern Blot. We are able to
“hunt” for a particular gene or DNA fragment by taking advantage of the structure of DNA.
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Dangerous Ideas and Forbidden Knowledge, Spring 2008
DNA is a double-stranded molecule, and the letters (bases) pair in a very specific way: A with T,
and C with G. Thus if I have a short sequence of single-stranded DNA reading AATGCA, the
only DNA molecule that can bind to it must read TTACGT. We will take advantage of this
specificity by creating a short single-stranded DNA molecule that can only bind to the
hemoglobin gene. This molecule is known as the probe. By artificially attaching a dye to this
probe, we will be able to find the sickle cell gene among the thousands of DNA fragments on our
membrane!
Part 1: DNA Electrophoresis
Note: We will work today in groups of 4-5 students, with each group running their own gel.
Make sure everyone has a chance to practice with the micropipettes and load a sample or two!
1. Each group will be provided with 6 tubes of digested (cut) DNA:
A = Sickle Cell gene sample
B = Sickle Cell Carrier sample
C = Normal gene sample
D = Mother’s DNA sample
E = Child’s DNA sample
F = Father’s DNA sample
Place all of your samples (with the lids tightly closed) in the 65 C waterbath for 2
minutes. Allow them to cool, briefly, before proceeding.
2. While your samples are heating and cooling, you can prepare by placing your gel, on its
tray, in the electrophoresis box. Be sure you place the wells of the gel on the black
(negative) end of the electrophoresis box. Carry your gel box to the front bench and
carefully add enough Running Buffer (1X TAE) to fill both end chambers and cover the
surface of the gel. Your gel is now ready to load.
3. Carefully adjust your smallest micropipette (2-20) to 20 uL. (It will read “2-0-0”.) Place
a clean tip on your micropipette and use your thumb to depress the plunger to the first
stopping point. Holding your thumb down, place the pipette tip below the surface of the
DNA sample and slowly release the plunger, drawing the DNA solution into your pipette
tip. Carefully position the pipette tip at the top of the first well in your gel and slowly
press down to the first stop, gently forcing the fluid out of your pipette tip. Pull the
pipette tip up away from the well before releasing your thumb.
4. Repeat step 2, loading each of the DNA samples into a different well on your gel, and
recording their position.
5. When you have finished loading your gel, place the cover on your electrophoresis box and
connect the box to one of the power supplies. Be sure to check that your DNA is loaded
on the negative end (black) as it will be drawn to the positive pole (red). We will let these
gels run at 110V for 30-40 minutes. Note that our DNA samples also contain one or
more negatively-charged blue dyes that will also migrate towards the positive pole.
While your gel is running:
With your gel up and running, let’s backtrack and have a look at these red blood cells.
With your team, obtain two microscopes and one each of the normal red blood cell smear
slides, and the sickle cell slides. Set-up each slide on the microscopes as you did last week.
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Dangerous Ideas and Forbidden Knowledge, Spring 2008
Have each team member examine both slides and record their observations on the worksheet
provided. Be sure to carefully, and correctly, put away both your microscope and the slides
you examine when you have finished.
When your gel is finished:
When your gel has been running for ~40 minutes, or when indicated by your instructor,
we will turn off the power supplies and begin our Southern Blots. Note that the loading dyes
will be visible on your gel, but that we cannot yet see the DNA. (DNA has no color, so we
will have to use another dye to make it visible at the end of our Southern Blot.)
1. Carefully transfer your gel (it will be wet and slippery!) to a plastic staining tray. Cover
the gel with 0.25N HCl. Note that this is a dilute acid, but should still be treated
cautiously. Allow your gel to soak for 8 minutes in this solution. If the dye turns yellow
before 8 minutes, proceed to step 2.
2. Carefully pour the HCl solution down the sink and rinse your gel with several changes of
water.
3. Now pour Denaturing Solution over the gel, completely covering its surface. Allow the
gel to sit in the Denaturing Solution for 15 minutes, periodically shaking the tray to
immerse the gel.
4. Pour off the Denaturing Solution and replace it with fresh Denaturing Solution. Allow the
gel to sit for 15 minutes. Do not discard this solution. Note that you can continue with
some of the following steps while your gel is soaking.
5. Obtain a tray from your instructor and place a piece of plastic wrap on it. Remove the gel
from the tray and place it, upside down, on the plastic wrap.
6. Obtain a piece of nylon membrane from your instructor, and cut it to match the size of
your gel. Take care to avoid getting fingerprints on the membrane.
7. Slightly bend the membrane in the middle and slowly wet the membrane (from the middle
out) in the Denaturing Solution contained in the tray from step 4. Release the membrane
and gently submerge it for 5 minutes until it is thoroughly saturated with the Denaturing
Solution. Place the saturated membrane on top of your gel.
8. Obtain a piece of filter paper from your instructor. Trim it to fit the size of your gel and
place it on top of the membrane, avoiding bubbles.
9. Carefully place a 4-5 cm stack of paper towels on top of the filter paper. Place your empty
tray on top of this stack. Place a 400 ml glass beaker, or something similar, on top of this
tray for weight.
The moisture from the gel will wick upwards through the membrane and paper towels,
carrying the DNA fragments with it. We will let this process run overnight. Someone will
then place your membrane in the fridge for you. We will return next week to try and find the
Sickle Cell Gene on our membranes!
Clean-up: As you leave the lab today, make sure your electrophoresis chambers and gel
trays have been rinsed with tap water, dried, and returned to their storage boxes. Place these
boxes on the carts in the classroom. Micropipettes and unused pipette tips should also be
returned to their boxes. Take care to wipe down your workspace if any solutions have been
spilled. The empty plastic tubes may be thrown in the trash.
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