Investigate Native Plant Evolution with Chloroplast Sequencing

Before beginning this science project, make sure you have all the necessary materials and equipment. You will also need to have done your background research and fully understand the principles behind PCR, gel electrophoresis, sequencing, and cladistics. The techniques in this science project are fairly advanced; the assumption is that you will either be familiar with using the PCR machine and gel electrophoresis equipment through your work in class, and/or will be using the equipment with a mentor. When in doubt, ask a teacher or mentor for advice.

Plant DNA Extraction

1. Gather healthy green leaves from three native plants. Identify each plant using field guides or by taking samples from known plants, such as those labeled at a nursery or botanical garden. If you're taking samples from a nursery or garden, make sure to obtain permission first.
   • Note: Although in the end only one plant sample can be sent for sequencing, we recommend that you start with three different types of plants in case one or more of them is so divergent in sequence that it fails to amplify using either primer set.
2. Give each plant a unique identification code. For example, if you gathered leaves from Ribes amarum (bitter gooseberry) and Quercus agrifolia (coast live oak) you could label the R. amarum "A" and the Q. agrifolia "B."
3. For each sample, pipette 200 µl of sterile double-distilled water (ddH₂O) into a PCR tube. Make sure to label each tube with the identification code of the plant sample you are going to place in it. For example, the tube containing R. amarum would be labeled "A."
4. Place the first plant sample on a clean white sheet of paper.
5. Punch out a small leaf disk by pressing the end of a sterile pipette tip into the plant leaf. The disk will be slightly larger than the pipette tip and will stick to the tip. If the plant is especially fibrous, you might have to apply pressure to the tip and pull the excess leaf tissue away from the pipette tip.
6. Let the leaf disk fall off the pipette tip into the water in the PCR tube. Because it's so small, make sure the disk is in the tube. Remember to double-check that the PCR tube is labeled properly.
7. Take a toothpick and guide the leaf disk to the bottom of the tube.
8. Grind the leaf disk into fine particles by twisting and turning the toothpick for 3-5 minutes. The water may turn a light green color and there may be pieces of tissue floating in the water.
9. Repeat steps 3-8 for the other two native plant leaves. Store the tubes of plant DNA in the refrigerator until you are ready to proceed to the PCR steps.

PCR of Rubisco Large Subunit

1. Label two PCR tubes per plant sample. One tube should be marked "1" for primer set #1 and the other should be marked "2" for primer set #2. For example, the tubes for R. amarum would read "A1" and "A2," while the Q. agrifolia tubes would read "B1" and "B2."
2. Pipette 7 µl of ddH₂O into each PCR tube.
3. Pipette 11 µl of Master Mix into each PCR tube.
4. To all PCR tubes marked "1," use a new pipette tip to add 2 µl of primer mix 1.
5. To all PCR tubes marked "2," use a new pipette tip to add 2 µl of primer mix 2.
6. Remove your plant DNA solutions from the refrigerator. Using a new pipette tip each time, add 2 µl of the plant DNA solution to each PCR tube. Pipette up and down 15-20 times to mix all the reagents. Check your labels carefully to make sure you are adding the DNA to the correspondingly labeled PCR tube.
7. Make up two negative controls; one control for each primer mix.
   1. These controls should contain 10 µl of Master Mix, 2 µl of primer mix, and 9 µl of ddH₂O. Do not add DNA. Remember to use a new pipette tip for each component.
   2. Label the tubes "neg1" and "neg2" for negative control of primer mix 1 and negative control of primer mix 2.
8. Place the PCR tubes (six containing plant DNA and two negative controls, for a total of eight tubes) in the thermocycler and use the following cycling parameters to amplify the rubisco large subunit:
   1. 94°C for 7 min
   2. 35 cycles of:
      • 94°C for 30 sec
      • 55°C for 30 sec
      • 72°C for 2 min
   3. 72°C for 7 min
   4. 4°C hold
9. Store the PCR products in the refrigerator until you are ready to run your gel.

Electrophoresis of PCR Products

You will need to use gel electrophoresis to visualize your PCR products in order to determine if the primers successfully amplified the rubisco large subunit from your plant DNA samples.

1. Make a 1.5 percent agarose gel (1.5 g of agarose for every 100 mL of TBE buffer). The exact volume of agarose solution you will need depends on the size of your electrophoresis gel box. Read the manual that accompanies the gel electrophoresis box to get a proper volume estimate.
2. While your gel is solidifying, prepare your samples for electrophoresis.
   1. Briefly centrifuge the eight tubes containing your PCR products to bring the liquid to the bottom of the tubes. If you do not have a centrifuge, gently tap the tubes on a countertop until all the liquid has collected at the bottom of the tubes.
   2. Take out eight clean PCR tubes (one for every PCR product, including the two negative controls) and carefully label them with your identification codes (for example, A1 for the R. amarum sample amplified with primer mix #1).
   3. Add 5 µl of loading dye to each of the newly labeled tubes.
   4. Using a new pipette tip for each sample, transfer 7 µl of each PCR product from the original PCR product tube to the corresponding newly labeled tube containing loading dye. Pipette up and down gently to mix the dye and PCR product. These tubes with loading dye + PCR product are your samples for electrophoresis.
   5. Return the original PCR product tubes to the refrigerator. Each tube should still contain approximately 15 µl of PCR product.
3. Load your loading dye + PCR product samples into the gel.
   1. In addition to your samples, you will want to load 5 µl of a 100-bp DNA ladder.
   2. Make sure to write down in your lab notebook which samples are loaded into which wells. You may find it easiest to place the 100-bp DNA ladder in one of the middle wells and run all the primer set #1 samples to the left of the ladder and all the primer set #2 samples to the right of the ladder.
   
   
   

   Diagram of samples in a gel. The gel includes seven samples in wells #1, 2, 4, 6, and 7 which are labeled A1, B1, 1Kb Molecular Marker, A2, and B2. Wells #3 and 5 are empty.

   
   Figure 1. The diagram shows an example of how to organize your samples in the gel.
4. When all the samples are loaded, attach the electrodes from the gel box to the power supply. Run the gel until the loading dye has migrated approximately halfway down the gel.
5. Once your samples have finished migrating, you will need to visualize the PCR products in the gel by staining and photographing the gel.
   1. Place the agarose gel in a staining tray.
   2. Pour enough ethidium bromide (0.5 µg/mL) to cover the gel. Wait 15 minutes. Caution: Ethidium bromide is a carcinogen. Always wear gloves and safety goggles when handling.
   3. Pour the ethidium bromide solution from the staining tray back into its storage bottle.
   4. Pour enough water into the staining tray to cover the gel. Wait 5 minutes.
   5. Pour the water out of the staining tray into a hazardous waste container and place the stained gel on a UV light box. Caution: Ultraviolet light can damage your eyes and skin. Always wear protective clothing and UV safety goggles when using a light box.
   6. Place the camera over the gel and take a photograph.
6. Analyze the photograph of your gel to determine which plant samples amplified well.
   1. If there was good amplification, you should see a single, strong band, approximately 200 base pairs (bp) in size. Both sets of primers result in roughly the same-size PCR product. Note: it is possible that only one set of primers works for a given plant.
   2. Choose only one PCR product which amplified well to send for sequencing.
   
   
   

   An agarose gel is stained and labeled with two primer sets separated by a 1 Kb molecular marker. Under ultraviolet light the samples and marker glow in a faint purple band and can be identified for sampling purposes.

   
   Figure 2. These samples show strong bands, which are approximately 200 bp in size, on the agarose gel. They are suitable for sequencing.

Sending Sample for Sequencing

1. Before beginning this science project, email Dr. Baysdorfer at California State University, East Bay (chris.baysdorfer@csueastbay.edu). Inform him that you are a student doing the Science Buddies Chloroplast Sequencing Project. Give him your best estimate of when you will be sending him your sample and ask him if that is a convenient time for him to sequence it. Remember, he is doing the sequencing on a volunteer basis and you may have to adjust your experimental timeline slightly to accommodate his schedule.
2. Once Dr. Baysdorfer gives you the okay, proceed with the experiment as outlined above and mail him the sample. (The sample is the half of the PCR product you have been keeping in the refrigerator.)
   1. Dr. Baysdorfer's reply will contain the address to which you should send the sample.
   2. Send the sample via overnight shipping (FedEx, Express Postal Service Mail, or a similar service). The sample will be okay out of the refrigerator for the 24-48 hours it takes to mail it.
   3. Clearly label the sample with species name, and which primer mix was used for the amplification.
   4. Dr. Baysdorfer can only accept one sample for sequencing.
3. When sequencing is complete, Dr. Baysdorfer will send you the sequence by e-mail.
4. Alternatively, if you have access to sequencing facilities through a mentor or a company that you can pay to sequence your samples, you may choose to do the sequencing yourself.
   1. If this is the case, there is no need to contact Dr. Baysdorfer.
   2. You may sequence as many samples as you see fit.

Analyzing the Sequence and Creating a Cladogram

1. Dr. Baysdorfer will send you a file, called a trace or chromatogram, which is the output from the sequencing machine. You will need to use a program to "read" the trace file and convert it to a file containing the DNA sequence.
   1. FinchTV is a free program used to view and convert chromatograms. It can be downloaded from: CNet.
   2. Once you have installed FinchTV, open the program. Drag and drop the chromatogram file that Dr. Baysdorfer sent you directly into the open window of the FinchTV program. You will see a series of spikes in multiple colors; this is the sequence data and each spike represents a nucleotide.
   3. To convert the chromatogram into DNA sequence. Go to File, and select "Export, DNA Sequence: FASTA." The resulting file contains the DNA sequence and can be opened with a word processor program.
2. Search for other plant rubisco sequences that closely match yours, using the BLAST (Basic Local Alignment Search Tool) from NCBI (the National Center for Biotechnology Information).
   1. Scroll down to the Basic Blast options and select "nucleotide blast."
      
      
      

      A screenshot shows the nucleotide blast option highlighted on the blast.ncbi.nlm.nih.gov homepage. The nucleotide blast option is located near the left-center of the screen and is the first link under the Basic BLAST header.

      
      Figure 3. The "nucleotide blast" option is highlighted in red.
   2. Paste your PCR product DNA sequence (which you converted from the chromatogram) into the top box in the section labeled "Enter accession number, gi, or FASTA sequence."
   3. In the section labeled "Choose Search Set" leave the "Human genomic + transcript" bubble filled in and select "Nucleotide collection (nr/nt)" from the drop-down menu. This option will widen your search to all nucleotide sequences in GenBank and other linked databases.
      
      
      

      Screenshot of the nucleotide BLAST search box on the ncbi.nlm.nih.gov website. At the top of the BLAST query search page there is a text box where users can fill in search terms. An option under the search box that allow for different databases to be searched is highlighted and other databases is selected (necleotide collection is selected from the drop-down list).

      
      Figure 4. The red text depicts where you should enter your sequence. The red circle highlights the correct database options.
   4. Leave all other search options on their default settings and click on the "BLAST" button at the bottom of the screen to start the search.
   5. The search results page will have two sections. The first is a graphic representation of how well the search result sequences match your input sequence. The second is a ranked list of matching sequences. In order to rank the matches according to which sequence has the closest nucleotide sequence to your PCR product, click on the "Max ident" column to rank the matches by maximum identity to your input.
      
      
      

      A cropped screenshot of the BLAST results page on the ncbi.nlm.nih.gov website. The results page in the BLAST tool on the NCBI webpage show results listed that provide additional information such as the percentage match a result has to the specific query string. To the right of the list a column labeled "Max Ident" is selected to sort the results by matching sequences.

      
      Figure 5. The red circle shows the location of the "Max ident" column.
   6. Scroll down the list. Are any of the matches from the same species as your PCR product? If not, congratulations! You're the first to sequence the rubisco large subunit from that plant! If you submit your sequence data to GenBank, it will be added to the public databases so scientists worldwide can access it and learn from it.

      Email to contact a Science Buddies scientist for help with submitting your sequence to GenBank.

3. Choose several of the BLAST matches to use in constructing a cladogram.
   1. Click on the accession code for the sequence you are interested in. This will take you to the report for that sequence.
      
      
      

      A cropped screenshot of accession codes listed on the BLAST results page on the ncbi.nlm.nih.gov website. The results page in the BLAST tool on the NCBI webpage show results listed with an accession code in the first column of every result. Clicking on the code will open a report for the specific sequence.

      
      Figure 6. The red circle shows an example of an accession code.
   2. Under the heading "Display," choose FASTA from the drop-down menu. FASTA is a text-based format for displaying sequence information. It is accepted by most other bioinformatics programs. Therefore, storing the sequences you are interested in with FASTA format is the best thing to do when you want to be able to use them with different bioinformatics programs later.
   3. From the "Send to" drop-down menu, choose "text." Copy the resulting FASTA-formatted sequence to a Word document for later use.
      
      
      

      A screenshot shows a nucleotide sequence displayed on a page hosted by NCBI. Above the sequence there is a display drop-down list where FASTA is selected and send to text is chosen as well. The FASTA sequence will be saved in a text document.

      
      Figure 7. This screenshot illustrates what a sequence looks like in FASTA format. The important options are highlighted in red circles.
   4. Repeat the procedure for as many sequences as you are interested in comparing. You may want to take a sampling of both closely matching and less similar sequences.
4. Using ClustalW2, build a cladogram of your PCR product sequence and the BLAST matches you've chosen.
   1. Go to the ClustalW2 homepage http://www.ebi.ac.uk/Tools/msa/clustalw2/
   2. Paste, in FASTA format, all the sequences you want to use to make your cladogram, including the sequence for your PCR product.
   3. Click on the "Run" button to obtain your ClustalW2 results. All the other parameters can be left on their default settings.
      
      
      

      A screenshot shows the ClustalW2 tool hosted on a webpage, there is a large textbox at the bottom of the homepage where FASTA sequences can be pasted into.

      
      Figure 8. Paste the FASTA-formatted sequences into the ClustalW2 tool, as shown in this diagram.
   4. The results page includes an alignment of all the sequences you inputted, and at the bottom of the page is the cladogram depicting the relationships between the different sequences.