Lab 2-2mtDNA Extraction and Amplification

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Dangerous Ideas and Forbidden Knowledge, Spring 2005
Lab 2
Part2: Analysis of mtDNA
Sequences
OBJECTIVES:
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Review the process of DNA replication, electrophoresis, and PCR
Understand the process of DNA sequencing
Explore the Bioserver database and Genbank
Compare and analyze our own mtDNA sequences
General Background:
Recall that earlier in the quarter we collected our cheek cells using a saline rinse, ruptured
those cells to extract their DNA, and then used the polymerase chain reaction (PCR) to make
multiple copies of a small portion of our mitochondrial DNA (mtDNA). (See Lab 2, Part 1 for
details.) When you last saw your sample, it was in the thermocycler, ready to begin that PCR.
In the time since, I have used DNA electrophoresis to visualize your samples. I took a
small portion of your PCR product (5 ul) and ran it on a gel to see if your reaction had worked. If
the PCR did not work, there was too little DNA to see on the gel. If it did work, a strong band
was visible on the gel. In this case, I then sent your PCR reactions to Cold Springs Harbor
Laboratory for sequencing on their DNA sequencers.
Cold Springs Harbor Lab technicians then used your PCR product as a template for DNA
sequencing, and visualized the results on an automated DNA sequencer. (See notes on
sequencing below.) The sequence they obtained has been posted on the Bioserver website. We
will access these sequences together in lab today and compare our mtDNA sequences to each
other, and to modern humans from around the world!
Notes on DNA Sequencing (also see figure at the end of this handout):
DNA sequencing takes advantage of what is known about DNA replication in cells. In
many ways, it is also quite similar to the reaction you performed, PCR, to copy your original
cheek cell mtDNA. As with PCR, heat is used to temporarily separate the two strands of a DNA
molecule. A DNA polymerase (the enzyme that copies DNA) can then use one strand as a
template to make a copy of the original molecule. When this reaction is done for the purposes of
PCR, it is done with a nearly unlimited supply of nucleotides (A, T, C, and G), the building
blocks of DNA.
In standard DNA sequencing however, this reaction is split into four separate tubes. Each
of these reaction tubes receives plenty of DNA polymerase and nucleotides, but also receives a
small amount of a modified nucleotide. (Thus one tube will receive a modified “A”, one tube a
modified “T”, one a “G”, and one a modified “C”.) This modified nucleotide (a
dideoxynucleotide) is unique in that it is unable to form a bond with the next nucleotide in the
growing chain. Thus these modified nucleotides are often called chain terminators. As the
polymerase moves along the template molecule, catalyzing the production of a new strand, it will
usually incorporate a “normal” nucleotide, but will occasionally incorporate a chain terminator.
When it does so, DNA replication stops. As many hundreds of thousands of these reactions are
occurring simultaneously in your tube, all possible lengths of DNA molecules will be produced.
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Dangerous Ideas and Forbidden Knowledge, Spring 2005
And in the tube with the modified “A”, all of these chains will end in “A”. This is true for the
tubes containing the T, G, and C chain terminators as well. Thus each tube contains a mixture of
molecules, all of which end in a particular nucleotide.
This collection of molecules is then sorted using electrophoresis. As with the
electrophoresis we did earlier this quarter, this process will sort the DNA molecules based on
their size. By running all four tubes next to each other, we can then “read” up the gel to
reconstruct the sequence of our original DNA template.
Common Questions:
1. Why didn’t my PCR work??
PCR is a notoriously finicky reaction. Common errors or sources of failure include
pipetting errors, and template quality. For example, if you had too few cheek cells in your
preparation, your PCR might not have worked. The presence of too many cheek cells, or other
contaminants, could also keep your reaction from working.
2. What does “N” mean in a DNA sequence?
Often times we cannot interpret which nucleotide (A, T, C, or G) is at a particular
location in a DNA molecule. When it cannot be determined, we insert an “N” into the sequence
to indicate an unknown nucleotide.
3. What makes some DNA sequences “excellent” and others “poor”?
On your data table, I have scored the results of each sequence on a qualitative scale
ranging from excellent to poor. This primarily reflects the number of N’s in your sequence.
Sources of sequence ambiguity can include poor template quality (PCR product) as well as
several factors out of your control, including the quality of the sequencing reaction and the skill
of the technician performing the sequencing!
Procedures:
Your goal today is primarily exploratory. You will work with one other student to access our
DNA sequences, practice some alignments, and generate phylogenetic trees from our sequences.
Students with good sequencing results may wish to identify their number, but note that this is
optional!
Step 1: To begin, open a browser to http://www.bioserver.org/sequences/.
the Sequence Server as a guest.
Login to
Step 2: Click the “Manage Groups” button at the top of the screen. This will open the Manage
Groups Window. In this window, choose “classes” from the popup menu on the upper right. A
new screen will appear and you will see our class listed under “Group 1- Murkowski” and “Group
2- Murkowski”. To select these classes, click the checkbox next to your class and click “OK”.
This will move our class onto your worksheet.
Step 3: To compare sequences, you will need to have more than once sequence on the worksheet.
To add students from our class, select the desired sequences from the popup menu. Then click
the check box for each sequence you want to include in your comparison, and press the
“Compare” button. Sequence server will open a new window to display the results of your
comparison. Note that sequences with many N’s (those rated “poor” on my table) are difficult for
the server to align!
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Dangerous Ideas and Forbidden Knowledge, Spring 2005
Step 4: Practice an alignment! Select two or more sequences for alignment as described in Step
3. In the space below, or on an additional sheet, make note of which sequences you choose to
align, and how many differences you observed between them. (Differences will be highlighted in
yellow; ambiguous positions are noted in grey.)
Step 5: From your main worksheet, you can also compare any of ours to those contained in the
international database, Genbank. To do this, select a sequence by clicking the round button to the
right of the sequence and then clicking “Analyze”. Sequence server will open a new window
showing the results of your analysis. Note that you can follow links (the Genbank accession
numbers) in the results to learn more about the sequences you match with. Try this with at least
one of our sequences.
Step 6: Now for the fun part! As we did last week, try using the Bioserver software to generate
at least one phylogenetic tree. From your main worksheet, return to “Manage Groups”. Notice
that you can add a variety of groups to your worksheet including modern humans, ancient humans
(those Neandertals!), other students, and other animals. Select two of more groups of interest to
you, and at least two of our students, and draw the phylogenetic tree you generate in the space
below.
Does the tree you’ve generated look like you would expect it to? Why or why not?
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Dangerous Ideas and Forbidden Knowledge, Spring 2005
DNA Sequencing (the Sanger Method):
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