Constructing a Phylogenetic Tree Using DNA Sequence Data

Mark Gabler

Target age or ability group: Second-year biology students or first-year students after a significant amount of background presentation and practice on the topic.
Class time required: Two class periods (45-55 mins. each), completing Part One the first day and Part Two the second.
Materials needed: (per lab group of 4-5) 4-5 Teacher-constructed DNA sequence strips (depending on size of groups) all groups start with the same sequence
One bag containing 100 pieces of paper, each numbered 1-100
One bag containing four pieces of paper, each with one of the letters A, G, C, or T on it
One standard die
Prior knowledge or concepts necessary to complete activity: It is expected that the students will have been introduced to evolutionary biology, as well as have some knowledge of DNA structure, function, and mutation.
Summary of activity: This activity is intended to give students an opportunity to analyze DNA sequence differences between organisms in order to establish a reasonable picture of the evolutionary relationships between them (Phylogenetic Tree). In simulation form they will be working through the same processes that molecular biologists use with the latest current technology. Equally important, prior to this simulation, the students will step through the evolutionary processes that lead to these DNA differences in organisms that once shared, in an ancestral organism, identical DNA sequences.

Additionally, though all groups begin the process with the same DNA sequence, they will each undoubtedly end up with very different descendant sequences. This can be focused on to demonstrate that if any evolutionary process were to "replay" itself, the ultimate results will never be the same twice. Evolution is not a planned event.

References: For more background information on this topic, you may wish to take a look at the following books.

Evolution at the Molecular Level, Selander, Clark, Whittam (eds.)

Fundamentals of Molecular Evolution, W.-H. Li and Dan Graur

Acknowledgment: I am grateful to Don Cronkite for his collaboration in the development of this module.

Constructing a Phylogenetic Tree Using DNA Sequence Data


To study the evolutionary relationships between organisms alive today, various methods can be employed to estimate when those organisms may have diverged from a common ancestor. Having this information for a group of living organisms would allow one to construct a phylogenetic tree. This tree depicts in graphic form these divergences over time and accounts for the current various species in question. Such a tree for six current species might look like the chart below:

In the past, much of this work was done by making observations of anatomy and physiology and with comparisons in fossil records. More recently, techniques have been developed in molecular biology for performing such comparisons. One of the newest and most quantitative methods is to compare the nucleotide sequence in a particular segment of DNA common to two or more living organisms. Often referred to as a molecular clock, this method utilizes a predictable rate at which mutations occur in DNA sequences. Those organisms that show the greatest number of nucleotide sequence differences are considered to have diverged from a common ancestor (following separate evolutionary paths) the greatest number of years ago (e.g., Nos. 1 and 6 in the diagram at the above). If two organisms have few nucleotide differences between them, but a large and approximately equal number of differences from some third organism, they would be closely related and likely be found on "twigs" of a branch that are far removed from that third species. (e.g., Nos. 3, 4, and 1).


In Part One of this activity you will be changing the "ancestral DNA" with random mutations over time, as well as making divergences into different evolutionary lines. As you do this, you will be constructing a phylogenetic tree that depicts these alterations. You will know that this tree is accurate because you have made it as you made the genetic changes. It's as if you�ve been around for millions of years recording all the changes that have gone on with the DNA of the ancestral form. You will end up with a unique nucleotide sequence for each of the present-day organisms.

In Part Two you will attempt to construct the phylogenetic tree of another group based strictly on the nucleotide sequences of their present-day organisms. This is what a molecular evolutionist would do.

Part One


  1. Divide your group into two new groups. Each of the new groups may be of any size.

  2. Each of these new groups gets a nucleotide sequence strip from the ancestral form. This marks the beginning of the first divergence, and the base of your phylogenetic tree.

  3. To determine how many base changes your group will make before the next divergence, roll the die and add 2 to the result.

  4. Each group randomly draws a number from the "Bag-O-Numbers," and a letter from the "Bag-O-Letters." The number indicates the nucleotide position from the left that gets changed and the letter indicates the base that it gets changed to.

    If the letter drawn matches the nucleotide in that position, draw again. It must cause a change. Return numbers and letters to their bag immediately.

  5. When you make a divergence (branch point), take another "fresh" ancestral DNA sequence, copy on to it the changes that have occurred to this point, divide the group into two parts, and continue as in step #3.Note how much time has elapsed since the last divergence based on the graph below. Try to scale your tree based on the passage of time.

  6. When the construction of the tree is finished, each person from the original group should represent one present-day organism (at the very tip of a branch). Put a different number on the back of each sequence with your group�s initials. Then put these numbers in their correct location on your tree.

    Rules for Part One

    • If you are by yourself (as a "group of one"), you may not make any divergences (this is only for the sake of simplicity). Otherwise you must make a divergence after the number of base changes indicated by the last roll of the die.

    • Be certain that no matter what path you follow from the original ancestor to a tip, the total number of base changes (mutations) must equal 25. So, toward the end of this "evolution game," either stop or continue making changes so that each DNA sequence ends up with 25 changes.

    Construct a graph that correlate numbers of base changes with the passage of time. This will help you to scale your phylogenetic tree both for time. For example, if the roll of the die says to make a divergence after six mutations, then that divergence occurs after approximately 15 million years.

    Part Two

    1. Now that you've finished your group�s Phylogenetic Tree, exchange your group�s DNA sequences with another group, and, working/thinking "backward" from what you just did, construct in the chart below their phylogenetic tree and label it, as you did yours, with the numbers on the back of their sequences.

    2. When you have finished this, have the other group check to see if your construction is closely similar to their tree. If it is not, think and analyze some more. If they approve of your phylogenetic interpretation of their DNA sequences, have a member of that group sign your work and then go on to answer the questions.

      Note: This is basically what a molecular biologist actually does to analyze DNA data from different present-day organisms in order to discover their evolutionary relatedness. Don't forget that you've got to compare each organism's sequence with all the others in order to draw valid conclusions.

    Constructing A Phylogenetic Tree Using DNA Sequence Data


    Build YOUR tree below:

    As you construct your tree, put in the correct time units along the vertical axis of this chart. Again, base this time on the graph on the previous page, depending upon how many base changes have been made since the last divergence.

    Build the OTHER GROUP'S tree below:

    Questions: Answer the following questions on your own sheet of paper and staple it to the back of this data page.

    1. Other than environmental effects (radiation, etc.), what causes base changes in DNA?

    2. Briefly state which step in the "scientific method" the phylogenetic tree represents AND WHY?

    3. Describe two ways in which other branches could be part of this tree but are just not shown?

    4. All groups began with the same base sequence. How many other sequences in class do you think are identical to yours? WHY?

    5. How many trees do you think are identical to yours? WHY?

    On to Using Amino Acid Sequences
    to Show Evolutionary Relationships

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