Download Dangerous Ideas and Forbidden Knowledge, Spring 2005 Lab 2

Dangerous Ideas and Forbidden Knowledge, Spring 2005
Lab 2
Part 1: Extraction and
Amplification of mtDNA
from Cheek Cells
OBJECTIVES:
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Become more familiar with cellular anatomy
Understand the general structure and function of DNA
Learn how to use micropipettes
Understand and perform the polymerase chain reaction (PCR)
General Background:
Last week in lab we viewed our own cheek cells, and noticed the large oval structure, the
nucleus, visible in the middle (roughly) of our cells. You probably are already familiar with a
key role of the nucleus; it is the location of most of your DNA. Your DNA contains the
directions for making all the proteins required to support life, carefully packaged on 46
chromosomes.
In addition to our very important nuclei, these cheek cells also contain many other
structures essential to their function. Among these other structures not so easily visualized are the
mitochondria. Mitochondria break down sugars and other energy-rich molecules to capture their
energy. This energy is then used by our cells for a variety of functions. Interestingly, our
mitochondria, like those of all plants, animals, and other eukaryotes, have their own DNA!
Stranger still, this DNA is dramatically different from that found in our nuclei.
Nuclear DNA is packaged on those 46 linear chromosomes. Mitochondrial DNA
(mtDNA) is packaged in a single, circular chromosome, and contains only 37 genes. (Your
nucleus, in contrast contains perhaps 30,000-50,000 genes.) Your nuclear DNA is acquired in a
very predictable manner, with half coming from your mother and half from your father. But your
mitochondrial DNA is all from your mother! It was present in mom’s egg and passed on to every
cell in your body during development. It is this unusual mitochondrial DNA that will be our
target today.
We’re after the mtDNA because it provides some powerful advantages for study. Each
of your cells contains hundreds of copies of mtDNA; this makes it easier to extract and isolate. In
addition, as your mtDNA is inherited from only your mother, we can use it to trace evolutionary
history without the complications of sexual recombination. This is our ultimate objective. We
will extract and copy our DNA in lab this week. Your samples will then be sent to Cold Springs
Harbor Laboratories for DNA sequencing. When they finish the sequencing, we will compare
our sequences to those of people from around the world!
Overview of Procedures:
We have two primary objectives in lab today:
1. Extract mtDNA- We will first need to collect some cheek cells. Once collected, we will
lyse these cells (break them open), to extract and purify our mtDNA.
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Dangerous Ideas and Forbidden Knowledge, Spring 2005
2. Generate copies of this mtDNA- Unfortunately we won’t be able to extract enough
mtDNA to be able to determine the sequence of bases (letters) in this informative
molecule. So we will need to make copies of the DNA we extract. We will do this
“copying” through the use of a common technique known as PCR (Polymerase Chain
Reaction).
The Polymerase Chain Reaction (PCR):
In 1983, Kary Mullis at Cetus Corporation developed
the molecular biology technique known as the polymerase
chain reaction (PCR). PCR revolutionized genetic research,
allowing scientists to easily amplify short specific regions
of DNA for a variety of purposes including gene mapping,
cloning, DNA sequencing and gene detection.
The objective of PCR is to produce a large amount of
DNA in a test tube starting from only a trace amount. A
researcher can take trace amounts of genomic DNA from a
drop of blood, a single hair follicle, or a cheek cell and
make enough to study. Prior to PCR, this would have been
impossible! This dramatic amplification is possible
because of the structure of DNA, and the way in which
cells naturally copy their own DNA.
DNA in our cells exists as a double-stranded molecule.
These two strands, or sequences of bases, bind to one
another in a very specific, predictable fashion. Specifically,
A’s will only pair with T’s, and C’s will only pair with G’s.
Thus if you know the sequence of one strand of DNA, you
can accurately predict the sequence of the other.
Both DNA replication and PCR take advantage of this predictability. In your cells, one
strand of DNA is used as a template to copy the sequence of your DNA from every time a cell
divides. PCR does essentially the same process, using one strand of your mtDNA as a template
to produce copies of its sequence.
PCR is conducted in three steps: 1) Denature the template DNA, 2) Allow the primers to
anneal, and 3) Extend (copy) the template DNA. In the first step, the template DNA is heated up
to break the hydrogen bonds holding the two strands together. This allows each strand to serve as
a template for generating copies of the DNA. In the second step, the temperature is reduced to
allow the primes to anneal, or bind, at their complimentary sequence on the template. (Primers
are short, specific pieces of single-stranded DNA that provide a starting point for the enzyme that
will do the ‘copying’.) In the third step, the temperature is raised again to allow the enzyme to
bind at the primer and add bases to the growing DNA molecule. These three steps are repeated
between 20 and 40 times.
The power of this process is that it results in exponential growth. After the first round of
copying, a single DNA molecule will have produced two identical copies. These two copies will
generate four molecules in the next round. Those four molecules will create eight, and so on.
Thus in 30 cycles we generate literally millions of copies of DNA from each template molecule,
plenty for DNA sequencing! (We will discuss the method of DNA sequencing later this quarter.)
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Dangerous Ideas and Forbidden Knowledge, Spring 2005
Part 1: Collection of Cheek Cells and Extraction of mtDNA
Note: Each student will be assigned a number in lab. This is the number you will use to label
your samples throughout our experiment! (So write it down someplace you can find it again.)
This number is used for ease of tracking samples and for confidentiality.
1. Use a permanent marker to place your assigned number on two clean 1.5 mL tubes, and on
the 15 mL test tube containing 10 mLs of saline solution.
2. Pour the saline solution into your mouth and vigorously rinse your mouth for 10 seconds.
Save the test tube for later use.
3. Expel the saline solution into the paper cup.
4. Carefully pour the saline solution from the cup back into the original tube, and close the
cap tightly. Save the cup for later use.
5. When we have all collected our samples, the instructor will place them in a clinical
centrifuge and spin the cells for 10 minutes.
6. After the spin, carefully pour the liquid into the paper cup. (This liquid can now be
disposed of by pouring it down the sink.) Be careful not to disturb the pellet of cells that
has formed at the bottom of the test tube.
7. Find your 1.5 mL tube containing the Chelax resin (labeled CR). Invert this tube several
times to mix its contents and pour them into the large tube containing your cell pellet.
(This resin will bind metal ions released from your cells as they rupture.)
8. Set your largest micropipette to 500 uL and place a clean tip on it. Gently pipette up and
down to thoroughly resuspend your cells. Then use your pipette to pull of 500 uL of this
mixed sample and transfer it to one of your clean, 1.5 mL tubes. Be sure each person in
your group uses a clean pipette tip for this step!
9.
Place your sample in a floating tube rack in the boiling water bath for 10 minutes. Do
not submerge or drop your tube in the water. The boiling temperature will break open
our cells, releasing their contents.
10. Use forceps to carefully remove your tube from the boiling water. Allow it to cool (on
ice if available) for 2 minutes.
11. Bring your tube to your instructor. She will place it in a microcentrifuge for a 30 second
spin.
12. Set your largest micropipette to 200 uL and place a clean tip on it. Use this tip to
carefully transfer 200 uL of the liquid from the top of your tube to your second, clean,
labeled tube. Be careful not to disturb the pellet of debris at the bottom of your tube.
Part 2: PCR amplification of mtDNA
1. Your instructor will provide you with a small PCR tube containing 22.5 uL of a PCR mix.
Label both the side and the top of this tube with your number. Note that these tubes have
very thin walls to facilitate heat transfer. Handle them gently!
2. In a separate tube, you will also be provided with a Ready-To-Go PCR bead. Carefully
transfer the bead to your labeled PCR tube.
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Dangerous Ideas and Forbidden Knowledge, Spring 2005
3. Select your smallest micropipette (2-20) and apply a clean tip. Use this fresh tip to add
2.5 uL of your DNA preparation to your PCR tube. Be sure to add your DNA solution to
the PCR mix (and not just the side of the tube), watching carefully as you pipette!
4. Place your labeled tube in the thermocycler. When all our samples are complete, your
instructor will program this instrument to heat and cool our samples, running them
through 30 cycles of PCR.
This cycling will take approximately three hours. When it’s complete, your instructors will
remove your samples, and send them off for DNA sequencing!
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