The Case of the Reclusive Relative Laboratory Procedure

Step 1) Background Theory

1. Go to the Dolan DNA Learning Center (http://www.dnalc.org). In the middle of the page you will see the following menu:

2. Click on 'Genetic Origins' and then choose the 'Mitochondrial Control Region' experiment. Read the "Theory" section. It has background information on mitochondrial DNA structure, mutations, and on how to use the region of the mitochondrial genome that we will be sequencing to infer evolutionary relationships.

Step 2) Isolating Cheek Cell DNA

1. Pour 10 ml of the saline solution (0.9% NaCl) into mouth and vigorously swish for 1 minute.

2. Expel saline solution into container provided.

3. Swirl to mix cells and transfer 1 ml (1000 L) of the liquid to a 1.5 ml tube.

4. Place your sample tube, together with other student samples, in a balanced configuration in a microcentrifuge, and spin for 1.5 minute.

5. Carefully pour off supernatant into paper cup or sink. Be careful not to disturb the cell pellet at the bottom of the test tube. A small amount of saline will remain in the tube.

6. Resuspend cells in remaining saline by pipetting in and out. (If needed, 30 L of saline solution may be added to faciliate resuspension.)

7. Withdraw 30 L of cell suspension, and add to tube containing 100 L of Chelex. Vortex to mix.

8. Boil cell sample for 10 minutes. Use boiling water bath, heat block, or program thermal cycler for 10minutes at 99°C. Then, cool tube briefly on ice (optional).

9. After boiling, vortex tube. Place in a balanced configuration in a microcentrifuge, and spin for 30 sec.

10. Transfer 30 L of supernatant (containing the DNA) to clean 1.5 ml tube. Avoid cell debris and Chelex beads. This sample will be used for setting up one or more PCR reactions.

11. Store your sample on ice or in the freezer until ready to begin Step 3.

Step 3) PCR Amplification of the Mitochondrial Control Region

1. Prepare the following reaction mixes on ice:

For a 25 l reaction volume:μComponent Volume Final Conc.To tube Containing PuReTaq Ready to go PCR Bead add: Mito forward primer 2.5 L μ 1.0 MμMito reverse primer 2.5 L μ 1.0 MμDNA template 5 L μ <250ngNuclease-Free Water 15 L μ

2. Mix by pipeting to dissolve bead.

3. Place the reactions in a thermal cycler that has been preheated to 95°C. Perform PCR using these parameters:

30 Cycles of:

Denature 94°C 30 sec.Anneal 58°C 30 sec.Polymerize 72°C 30 sec.

Hold: 4°C Overnight

Step 4) Quantifying and Analyzing the PCR Products

The sequencing reactions must be performed on carefully quantified samples that only contain a single amplified fragment of the correct length. We will quantify and analyze your PCR products in two ways.

Analyzing PCR Amplification Specificity and Fragment Length using Agarose Gel Electrophoresis.

1. Add 50 mL of 1XTAE (Tris Acetate EDTA) buffer to a 125 mL Erlenmeyer flask for the small gels and 100 mL 1X TAE buffer to a 250 mL Erlenmeyer flask for the large gels.

2. Weigh out 0.5 g of agarose (small gel) or 1.0 g of agarose (large gel) and add it to the 1XTAE.

3. Microwave the mixture carefully, removing the flask from the microwave and swirling gently each time you see the mixture start to boil. When the bubbling subsides, place the mixture back in the microwave and continue the process until all of the agarose has melted. This will probably take less than 2 minutes for the small gels, and between 2-3 minutes for the large gels.

Safety Note!!

These agarose mixtures have a habit of superheating, so they will go from not boiling at all to an almost explosive boil when you begin to swirl them. Do not point the flask mouth at your own face, your lab partner’s face, or anything else that should not be covered with boiling, molten agarose.

4. Set up an agarose gel chamber by placing the gel mold sideways in the chamber (make sure the rubber gaskets stay in their channels to form a good seal). Wetting the rubber gaskets at this stage may help with the process. Next, place a comb in the gel mold and pour the molten agarose into the mold.

Rinse the flask immediately with lots of water to remove residual agarose.

5. Allow the gel to solidify for 40-60 minutes. While the gel solidifies, perform the Nanodrop analysis below.

6. After the gel has hardened, combine 2 L of your PCR reaction with 1 L of EZ Vision stain in a clean tube.

7. Load entire mixture into the well of an agarose gel. Your teammates should also load their samples into adjacent wells. Finally, load the DNA size standard sample that your instructor will provide into the final well. Run the gel for 20 minutes at 100 volts.

8. Visualize your samples using the transilluminator and take a picture of the gel.

9. Estimate your fragment concentration using the picture of your gel and the key to the concentration of each fragment below.

10. Is your fragment the correct length?

Determining DNA Concentration Using the Nanodrop System:

1. Log on to the computer in the molecular research lab.

2. Turn on the Nanodrop program, and choose “Nucleic Acid” from the menu of options.

3. Place 2 L of ultraclean water on the pedestal in order to initialize the instrument.

4. Use the same water drop to blank the instrument by clicking on the ‘Blank’ buttion.

5. Verify that the water drop has a concentration of essentially 0 ng/L by clicking on ‘Measure.’

6. Use a Kimwipe to remove the water droplet, and carefully place 2 L of your sample on the pedestal, making sure not to generate any air bubbles. Click on ‘measure’.

7. Record your concentration in ng/L .

Step 5) Sequencing your Mitochondrial DNA

Your samples will now be mailed to GenScript, a commercial DNA sequencing facility. They use Sanger DNA sequencing, which is good for relatively short templates, such as ours. We will have our sequences back in just a couple of days. Most of that time, by the way, is shipping. For entire genomes, folks now use new technologies called “Next Generation” sequencing that can allow you to sequence an entire genome in a matter of days.

Step 6) Analyzing your Mitochondrial DNA Sequences

Trimming your mitochondrial DNA sequence using 4Peaks.

In order to determine how related you are to your classmates, to Rarely Reclusive, and to the human ancient and modern groups in the Dolan DNA database, you will need to first determine whether or not your sequence is of sufficient quality for the analysis, and then to trim your sequence so that only good quality data is used. The 4Peaks program that you used during your yeast experiments will work very well for this purpose.

1. Examining the primary data of the mitochondrial control region

Open up the file labeled “mito(x).scf” using the program “4Peaks”, where (x) is the identifier for your mitochondrial DNA sequence (the ). To do this double-click on the “4Peaks” icon, and under the pull down heading of “File” select “Open”. Then select the file name “mito(x).scf” from the “mitochondrial DNA sequences” desktop folder. This computer file contains the sequence data we obtained from the mitochondrial control region of your mitochondrial DNA. At this point you should see peaks of four different colors. There is also an X-axis that consists of a time unit of measurement, a nucleotide number, and a nucleotide (designated A, C, G, or T). There are a few things to note:

i) at many points there is only one peak and that the color of the peak determines the nucleotide indicated on the X-axis. The sequence at these points is of high quality, and can be used for phylogenetic analysis. A sample is shown below:

ii) at some points, there is more than one peak (see bases near the beginning, for example). This is due to technical problems and makes it difficult or impossible to predict the base that is at that position on the DNA strand. This is why there is occasionally a “N” instead of an A, C, G, or T. “N” designates that the identity of the base at this position was not clearly determined. Sequence with too many “Ns” cannot be used in phylogenetic analysis. An example is shown below, although some sequences will certainly look even worse than this:

iii) Look at your sequence. Does it have a section with good sequence, or are there N’s all the way through it? If there is good sequence available, highlight (select) the good sequence by clicking on the first base of the good stretch, and then holding and dragging to the right until you have selected the last base of the good stretch. If your sequence is not of sufficient quality, do not worry. Simply work with a sequence from your team that is.

iv) Select "Crop" from the drop-down menu in the lower left hand corner of the window (the ‘gear’ icon). The sequence that remains in the 4Peaks window is now just the good quality DNA. Use “Export” to rename this cropped file

and to save it to the desktop. Change the .txt to .fas This step must be done or the MEGA 5 program will not recognize the sequences.

v) BLAST Analysis of your cropped sequence Under “Edit” choose “BLAST Sequence” then Nucleotide

(BLASTn). This is the same program that you used for your yeast DNA sequence comparisons.

You should see your sequence in the query window. Click on “BLAST”.

Does your sequence appear to be human mitochondrial DNA? How do you know? What is the percent coverage, the percent identity and the E value? What do these values mean?

vi) Repeat this process for all of the members of your team.

Step 7) Phylogenetic Analysis of the mitochondrial control region Now it is time to fine out how closely related you are to rarely reclusive, to other members of your team, and to students from last year. You will do this using the MEGA 5 program.

i) Open MEGA 5 by double-clicking on its icon. You will see a window that looks like this:

ii) Begin your analysis by clicking on “Align” and then clicking on “Edit/Build Alignment” from the drop-down menu. Choose “Create a New Alignment” and click ‘Ok’, and then “DNA”. You should see a window that looks like this:

iii) Import your sequences. Under “Edit” choose ‘Insert Sequence From File”. Make sure that the ‘Select Files of Type’ window at the bottom shows “FASTA”. Select your sequence, and click on “Open”. Your sequence should now appear in the Alignment Explorer Window. Repeat this step for each sequence from your team, and for Rarely Reclusive (Rarely.fas, respectively). As you import each sequence, you will notice that it comes in ‘selected’ (all blue). You can unselect (and reselect) the sequence by clicking on the sequence name to the left. You should notice that each base is a different color when the sequences are not selected.

“Aligning” sequences makes sure that you are comparing the sequences of the same parts of a gene with each other. The sequences above are not aligned (yet), so even though they are all mitochondrial DNA, they do not look like they match. When you ask MEGA to align sequences, it will search for large areas of identity and similarity, and then slide the genes around until those areas are matched to each other. The match will never be perfect (unless you have two identical sequences, of course), so the program is really looking for the ‘best match’.

iv) Align your sequences by selecting all four sequences (shift click), and then clicking on ‘Alignment’ and “Align by Clustal W” in the Alignment Explorer window. Click “Ok” to accept the default parameters. When you align your sequences, you should now see some of them match pretty well, while others may not. The computer will also insert short gaps into the sequences if it needs to to make things align better. In the aligned sequences below, the first three sequences are identical, while the next two are aligned, but are obviously NOT perfect matches.

So, who is most closely related to Rarely Reclusive? You will answer this question using UPGMA analysis. As we discussed in class, this analysis not only looks at the total number of nucleotide differences between individuals, but it also maximizes the parsimony of the tree. You will notice as you play around with MEGA that there is a specific “Maximum Parsimony” phylogeny program. However, in this analysis, we do not want to penalize sequences for being of different lengths. Remember that each of you trimmed your sequences to different lengths based on sequence quality, not based on evolution, we do NOT want the program thinking length differences are significant! UPGMA analysis will only use the bases that all of the sequences have in common when it constructs the tree.

v) Go Back to the MEGA 5.05 Window and click on “Phylogeny” and then “Construct/Test UPGMA Tree” and “Yes” to use the alignment file that you are currently working on, and then click on “Compute”. You should now see a phylogenetic tree in a window that looks something like this:

You can save the file as a .tiff image under the “Image” menu. The image can then be pasted into your Word document for your final lab report (question 2).

Which student in your analysis is most closely related to Rarely Reclusive?

Step 7) Phylogenetic Analysis – Human evolution and your place in the human family.

Well, you can't all be most closely related to Rarely, so who are you related to?

1. Follow this link (http://www.bioservers.org/bioserver/) to the Dolan DNA Bioserver login page. Create a username, a password, and log in to the Sequence Server site.

2. Click on 'Manage Groups' from the menu in the center of the top of the page. You will now see a window that looks like this:

3. Click on the upper right pull-down menu (sequence sources). You should now see a variety of choices. Choose 'modern human mt DNA'. Click on the boxes to the left of each of the sequences to select them, and then click on 'ok'.

4. Click on 'Manage Groups' again. This time select 'Prehistoric Human mt DNA' and click on all of the boxes, and then 'ok'. You have now uploaded a whole bunch of mtDNA sequences for analysis.

5. To upload your own sequence, go to your FASTA sequence, select only the sequence itself, and copy it.

6. In the sequence server window, choose 'Create Sequence'.Give your sequence a name, and then paste your sequence data into the window. Click 'ok'.

7. You will now see your sequence added to the rest. Do the same thing for all of your group members.

8. To compare your group members to the available sequences, click on the box to the left of your sequences, and then next to any other sequences that interest you (you'll see lots of drop-down possibilities within each group). You may only select up to ten total sequences (including your group sequences). Have fun, but also use sequences that make sense, given what you know about your ancestry.

9. After you have selected 10 sequences for analysis, find the word 'compare' in the gray bar menu, and choose 'phylogenetic tree' from the drop-down box. Click on "Compare". After your sequences are analyzed, a popup window will show you your tree.

10. Choose 'phenogram' and 'yes' (to make the tree branch lengths proportional to the evolutionary distances). You will see something that looks like the picture below, but that contains the individuals and species that you chose to analyze.

11. Phenograms such as this one can provide a ton of information. For example, one thing that this phenogram shows is that Lake Mungo Man and African American #1 share a common ancestor, to which Lake Mungo Man is more closely related.

12. Use the Grab program (scissors on the dock) and then choose “Capture” and “selection.” Select the phylogenetic tree by drawing a box around it and releasing the mouse. You will now see a window with the tree image in it. Next click on ‘copy’ to copy the image, and then go to your lab report and paste the image into the report (question #3).

Genetics Final Lab Report, Fall 2012. Bioinformatics and Human Evolution

Names:

1) In the spaces below, type the BLAST Values for your team’s mitochondrial DNA sequence(s) and provide a brief description, in your own words, of what those values mean in terms of your sequence matches with human mitochondrial DNA.

% Coverage:

E value:

% Identity:

2) Paste your MEGA 5 Phylogenetic Tree in the space below. Which classmate is most closely related to Rarely Reclusive? Justify your answer.

3) Paste your Phylogenetic Tree from the Dolan DNA Server in the space below. Describe, in your own words, the evolutionary relationships shown in the tree.