Comparing mtDNA Sequences to Learn about Human Variation
Developed by Scott Bronson

Grade Levels: 9-14
Subject Matter: Biology, Physical Anthropology, Genetics
Time Allotment: 2-3 class sessions
Description: This computer-based lesson will enable students to test their notions of "racial" similarity and difference by comparing mtDNA sequences as the students do in the first episode of RACE - The Power of an Illusion. Students can either sequence and compare their own mtDNA (with each other as well as with individuals from around the world) or compare public sequence files from different world populations to gain an understanding of human genetic variation.

Scott Bronson is Education Manager at the Dolan DNA Learning Center of Cold Spring Harbor Laboratory in New York. He is featured in Episode 1 of the series.

OVERVIEW

We can explore our species' shared ancestry - and what it tells us about the concept of "race" - by deciphering the DNA sequences of people from around the world. Are the different "races" of humans genetically distinct? Or is there just as much genetic diversity within so-called races as there is between them? Is race better defined as a social concept or a genetic concept? The computer-based activities in this lesson will help address these questions.

Is race genetically meaningful?

If race were genetically meaningful, most genetic diversity would be found between two races rather than within a race. In other words, members of one so-called race would be more similar to each other than to members of another so-called race. That means that one would expect to find more genetic differences between a random person from Africa and a random person from Europe than between two random people from within Africa or two random people within Europe. But is this really how people sort out?

In this lesson, students will collect and examine genetic data to better understand human variation from a geneticist's point of view. They will analyze and compare mitochondrial DNA sequence diversity within and between "races" to determine whether or not what we call races are genetically distinct. They will then explore the implications of their findings for their understanding of race.

How do you measure genetic distinctiveness?

In order for populations to evolve into distinct subspecies (the taxonomic category that corresponds to races), they must be isolated from one another long enough for sufficient DNA mutations to accumulate in one population to differentiate them from other populations. One way to find out is to examine sequence differences that have accumulated in our mitochondrial DNA (mtDNA).

MtDNA is a special type of DNA found in the cellular organelle called the mitochondrion. Mitochondria are responsible for producing energy, earning them a description as the "powerhouse" of the cell. Unlike DNA that is inherited from both parents, mtDNA is inherited only from the mother. Over time, mutations accumulate as mtDNA is passed essentially unchanged from one generation to the next, leaving a genetic "footprint" of one's mother's mother's mother's mother, etc. These accumulated mutations can be seen as mtDNA base sequence differences in people around the world.

Scientists approximate the evolutionary time back to the "common ancestor" of two different mtDNA sequences by assuming an average rate of mutation over the millennia. In this way, some scientists have calculated that all extant mtDNA sequences can trace their ancestry to a single woman in East Africa approximately 180,000 years ago. She is referred to as the Most Recent Common Maternal Ancestor - or simply Mitochondrial Eve. (That's not to say that all humans descended from this Eve; just that hers is the only mitochrondrial line to survive.)

NOTE: This lesson may challenge and confuse students, confronting as it does preconceived notions of biological race and thus, for some, self-identity. A discussion of race as a social concept (drawing upon other excerpts from RACE - The Power of an Illusion) may help address some of these issues. In any case, teachers should be sensitive to students' reactions and guide them appropriately.

OBJECTIVES

1. Students will explore patterns of human genetic variation.
2. Students will learn about mtDNA and the molecular clock.
3. Students will learn how to compare mtDNA sequence data from diverse human populations around the world.
4. Students will understand what mtDNA sequence information tells us about patterns of human variation and the concept of race.
5. Students will learn how our history of migration and gene flow has resulted in most human variation being within groups rather than between groups.
6. Students will contribute to a long-term project about global human genetic variation.

MATERIALS

• RACE - The Power of an Illusion Episode 1: The Difference Between Us (video or DVD)
• World map in the classroom
• Computers (one for every 2-3 students) with Web access to Cold Spring Harbor's Dolan DNA Learning Center Sequence Server
• Computer Projector
• Calculators (optional)

GLOSSARY
mtDNA
polymorphism
single-nucleotide polymorphism (SNP)
base-pair
mitochondria
mutation
subspecies

PROCEDURE

Depending on time and resources, your students can sequence and compare their own mtDNA with each other and then with populations around the world, as the students do in the film, or they can skip their own sequencing and just compare existing mtDNA sequences from around the world.

A note regarding sequencing your students' mtDNA: You can have your students' mtDNA sequenced free of charge through Cold Spring Harbor's Dolan DNA Learning Center. Click here for basic instructions. Getting the results takes 10 business days, so be sure to schedule the sampling several weeks prior to the unit. Additional information and support curriculum for analyzing mtDNA can be found at the Genetic Origins Web site.

A note on using video clips: This exercise intersperses several clips from Episode 1 of RACE - The Power of an Illusion. Some teachers may prefer to play the entire episode prior to the exercise. If so, consider returning or at least referring to the indicated clips to recapitulate themes. A complete transcript of Episode 1 can be found here.

LESSONS

ACTIVITY #1 - Articulating Student Preconceptions about Race

It's helpful to have students voice their own beliefs about race and genetics at the beginning of this lesson. Prior to the lesson, students should already understand the difference between genotype and phenotype and how human genetic variation results from mutations accumulating in DNA over time. Students should also know what mtDNA is and why it is useful to geneticists. Students should read about "Maternal Inheritance of Mitochondrial DNA" and "The Molecular Clock and Anthropology" at the Genetic Origins Web site (pages 5 and 6 of the Web text) before performing this activity.

Tell your students they will be sampling their own mtDNA and / or comparing mtDNA sequences between individuals from populations around the world to look for genetic differences (polymorphisms). But first ask them to write down the names of the class members they feel they will be most similar to genetically and most different from. Then, as a class, elicit definitions of race. Discuss what genetic criteria human populations would have to meet to be considered separate races.

Show the students two video clips from RACE - The Power of an Illusion Episode 1 - The Difference Between Us (8 minutes total):

CLIP A: Sets up the idea of race and the students' preconceptions about similarity and difference. Begins with the opening of the film through Alan Goodman talking about a paradigm shift. (00:45 - 05:25; DVD Scene #2)

CLIP B: Describes mtDNA and shows the students using computers to compare their mtDNA base-pair sequences. Begins with Alan Goodman talking about non-concordance and ends with the narrator concluding that the students didn't find racial matches in their mtDNA. (32:20 - 35:56; DVD Scene #10)

ACTIVITY #2 - Comparing Public mtDNA Sequence Samples from World Populations

Explain to the students that they will be performing similar comparisons of mtDNA taken from people around the world. Students will use the Sequence Server Web site of Cold Spring Harbor's Dolan DNA Learning Center for this activity.

NOTE: For the purpose of this exercise, it is assumed that "race," should the concept be valid, would correspond with continental origin.

First, you will demonstrate how the Web site works. At this point, it is very helpful to have the ability to project the computer screen in the classroom.

1. Navigate to the Sequence Server Web site located at http://www.bioservers.org/html/sequences/sequences.html.
2. Click the button to LOGIN AS GUEST. This will give you access to the server.
3. Click "Manage Groups" near the top of the screen. This will open a new window with folders of mtDNA sequences from high school classes around the country.
4. Click on the tool bar labeled "Sequence Sources." You should see other types of DNA sequence groups in the pull-down menu.
5. Find "Public" on the list. Highlight this group with your mouse to select it. This will pull up a list of groups.
6. Scroll down until you find "Race - lesson 2". To select this group of mtDNA base-pair sequences, check the box to the left of the group, and click the "OK" button. This will bring sequences from this group into your workspace in the main window. By clicking on the tool bar, you should see 9 mtDNA sequence files from different parts of the world. At the top of the list is the mtDNA sequence file called "Africa #1." Notice that the computer automatically checks the box to the left of the sequence file. The box must be checked to select this sequence for comparison.
7. After choosing your first sequence, a second pull-down menu will appear underneath. Scroll down until you see a sequence file called "Africa #2." Click on it to choose it. Notice that the computer automatically checks the box to the left. This box must be checked to select this sequence for comparison.
8. Compare the two sequences by clicking the "Compare" button in the upper left corner of the page. (When you click on "COMPARE", the computer uses a program called CLUSTALW to align the samples where they have similarities.) Differences will appear highlighted in yellow.

NOTE: If you are having trouble with the Sequence Server, try running this compatibility wizard to make sure that your browser is set up to work correctly with the server: http://www.bioservers.org/bioserver/cwizard.html. (Follow the directions on the screen.) If you continue to have problems, contact dnalc@cshl.org. Be sure to mention that you are using the Sequence Server.

Now that the students understand how the computer tool works, have them break into small groups and assign each group one of the following comparisons to perform:

• African mtDNA with African mtDNA
• European mtDNA with European mtDNA
• Asian mtDNA with Asian mtDNA
• African mtDNA with European mtDNA
• European mtDNA with Asian mtDNA
• African mtDNA with Asian mtDNA

Instruct the students to perform all of the possible base-pair sequence comparisons for their assigned populations. It is critical that students only compare 2 samples at a time, as multiple sequence alignments take longer to analyze. Have them count the number of base differences for each sequence comparison and then calculate that result in terms of a percentage. The students should write their results next to an assigned comparison chart on the board for the class to see. They should also calculate the average of their results and write that figure in the final column. This will allow students to observe variation within and between the races.

SAMPLE CHART

Comparison Group	Base-Pair Differences (by number and %)
	Indiv 1 & 2	Indiv 1 & 3	Indiv 2 & 3	average
African mtDNA & African mtDNA
European mtDNA & European mtDNA
Asian mtDNA & Asian mtDNA
African mtDNA & European mtDNA
European mtDNA & Asian mtDNA
Asian mtDNA & African mtDNA

After all the results have been posted, ask students to discuss within their small groups the following questions:

• Were the results surprising? If so, why?
• Was there more genetic diversity within or between continents?
• On which continent was the most diversity found?
• What might the results of these mtDNA comparisons tell us about human origins and the concept of race?
• Do they support the idea that human races are genetically distinct?
• How might the evidence lead you to redefine race as a concept?

ACTIVITY #3 - Sequencing and Comparing Your Students' Own mtDNA

If you've sequenced your own students' mtDNA, your students can align and compare their mtDTA sequences with the mtDNA sequences of their classmates, once you've been informed that your sequences have been posted on the Sequence Server.

Rather than compare all the sequences in the class against each other, have the students break into small groups of four or five to calculate the base-pair differences within their group. Each group should include a diverse mix of students, including those who, as indicated in Activity 1, believed they would be similar to each other and those they assumed would be different from them.

 

INSTRUCTIONS FOR COMPARING mtDNA SEQUENCES - HANDOUT Navigate to the Sequence Server Web site located at http://www.bioservers.org/html/sequences/sequences.html. Click the button to LOGIN AS GUEST. This will give you access to the server. Click "Manage Groups" near the top of the page. This will open up a new window with folders of mtDNA sequences from high school classes around the country. Scroll down until you find your class. Check the box on the left and click "OK." This will bring sequences from your class into your workspace in the main window. By clicking on the tool bar, you should see mtDNA sequence files for each student. At the top of the list you will see a file entitled "Student #1." Notice that the computer automatically checks the box to the left. The box must be checked for the file to be selected. Scroll down until you find the sequence file for the first student you would like to compare. Check the box and click on the file to select. After selecting your first sequence file, a second pull-down menu will appear below. Scroll down the page until you find the second sequence file you would like to compare (e.g., "Student #2") and click on it to select. Notice that the computer automatically checks the box to the left of the file. The box must be checked for the file to be selected. Compare the two sequences by clicking the "COMPARE" button at the upper left of the screen. (When you click on "COMPARE", the computer uses a program called CLUSTALW to align the samples where they have similarities.) Differences will appear highlighted in yellow. Count the number of differences and calculate the result as a percentage. Record your results in a chart for your group.

Each group should complete a chart with their findings as follows:

(sample chart)

Comparison Group	# Differences	% Difference
Student A / Student B
Student A / Student C
Student A / Student D
Student A / Student E
Student B / Student C
Student B / Student D
Student B / Student E
Student C / Student D
Student C / Student E
Student D / Student E

When everyone is finished, each group should report its findings to the class as a whole.

ACTIVITY #4 - What Does It All Mean?

Debrief with students about the results of their sequence comparisons: What was surprising? Did their results confirm their original expectations? What did they learn? Did the idea of human races hold up? Why do they think they got the results they did? What are the strengths and weaknesses of a genetic approach to the question of race?

To deepen their understanding further, show the following clips from RACE - The Power of an Illusion, Episode 1 (10 minutes):

CLIP C: Students compare their own mtDNA sequences to a worldwide database. Begins with teacher Scott Bronson and ends with the narrator explaining that today's findings corroborate Lewontin's discoveries. (45:17 - 49:47; DVD Scenes 15 & 6)

CLIP D: Describes Richard Lewontin's discovery that most human genetic variation falls within, not between, groups. (35:56 - 38:18; DVD Scene 11)

CLIP E: Human groups haven't been around long enough or isolated long enough to evolve into races. Begins with narrator saying "Race does not account for patterns of genetic variation" and ends with Stephen Jay Gould explaining there just hasn't been enough time. (40:46 - 43:33; DVD Scene 13)

NOTE: Some teachers may prefer to play CLIP C at the beginning of the lesson along with CLIPS A & B rather than at this point.

Following the clips, review the concept of within-group vs. between-group variation and how that helps explains the students' findings: On average, how much variation falls within vs. between local populations? Why does the disproportionate amount of within-group variation show us that biological race is a fallacy? Think about human history - what is it about human history that has resulted in so much human variation falling within groups and so little between groups? Would you expect to find similar results in other mammals, such as chimpanzees, chipmunks, and elephants? Why or why not? (For more information, read the "Go Deeper" associated with the "Human Diversity" portion of the RACE companion Web site.)

Students can explore these and other scientific concepts further in the "Human Diversity" section of the RACE companion Web site.

EXTENSIONS

1. How does your mtDNA compare to individuals from around the world?

If your students have sequenced their own mtDNA they can work in small groups to compare their own sequences to individuals from populations around the world, just like the students did in Episode 1 of RACE - The Power of an Illusion. This is done by performing a search of Genbank:

1. Follow the instructions above to bring mtDNA sequences from your class into your workspace.
2. Click the "Manage Groups" button at the top center of the screen. This will open up a new window containing folders of mtDNA sequences from high school classes around the country.
3. Click on the tool bar labeled "sequence sources" to find other types of DNA sequence groups.
4. Scroll down until you find the "Modern Human mtDNA" sequence source. Highlight the group with your mouse to select it. This will pull up a list of folders from different populations.
5. Scroll down the screen until you find a population you would like to work with. Choose the group by checking the box to the left and clicking on "OK." This will bring sequences from this group into your workspace.
6. In the pull-down menu, select a sequence file to work with from the chosen population (e.g., mtDNA sequence file #1). Select a sequence file from within your class for comparison, and click "COMPARE" to see the results. The computer will align the samples where they have similarities and highlight differences in yellow.
7. Count the differences and calculate the result as a percentage.

Have each group report their discoveries to the class. What surprising similarities and differences did they find?

2. Can We Determine an Individual's Race from His or Her DNA?

Students can analyze mtDNA sequences from four individuals whose race is unknown.

Tell your students that 3 of the mtDNA sequences are from African individuals and one mtDNA sequence is from a European, and ask them if they can determine which is the European:

1. These sequences are found in a folder in the "Public" section of the Sequence Server.
2. Click "Manage Groups" at the top of the screen. This opens up a new window with folders of mtDNA sequences from high school classes around the country.
3. Click on "sequence sources" to see other types of DNA sequence groups.
4. Scroll down to the folder labeled "Public." Highlight this group with your mouse and click to choose it.
5. Scroll down the page until you find the folder titled "Race-lesson 2 extension." Select this folder.

Working in small groups, have students attempt to determine which of the mtDNA sequences is European. Have each group share with the class which sample they believe is European, then reveal the identity of all four samples with the students. (The European mtDNA is sequence #3.) Ask the students to explain why it is difficult to determine "race" at this genetic marker.

ASSESSMENT

Final assessment can be based on a written report of the exercise. Students should begin the report by discussing who they originally thought they'd be most similar to/different from and why; what they found in their exercise; and what they learned about human genetic diversity. They should also discuss how their view of the "race" concept has or has not changed as a result of this exercise. The following topics should also addressed in their report: do these mtDNA comparisons support the idea that race is a genetically valid concept? Why or why not? What are the strengths and weaknesses of using mtDNA to understand human genetic relationships and race? If not by race, how would they describe patterns of human variation? The report can be evaluated on how well students use the mtDNA evidence and information from the film to support their positions.

ADDITIONAL RESOURCES

RACE - The Power of an Illusion companion Web site:

• Explore Human Diversity
• Background Readings - Science
• Ask the Experts Science

The Genetic Origins Web site: http://www.geneticorigins.org/geneticorigins/

Human Origins and Evolution in Africa: http://www.indiana.edu/~origins

Larry Adelman, "Race and Gene Studies: What Difference Makes a Difference?"

Roger Lewin, "The Molecular Evolutionary Clock" in Patterns in Evolution: The New Molecular View. Scientific American Library, New York: 1997, 1999.

Richard Lewontin, Human Diversity. Scientific American Library, New York: 1982, 1995

Steve Olson, Mapping Human History: Discovering the Past Through Our Genes. Houghton Mifflin, New York: 2002

RELEVANT STANDARDS

From Mid-Continent Research for Learning and Education at http://www.mcrel.org/

Life Sciences Standard 4 Level IV (Grades 9-12):

1. Knows ways in which genes (segments of DNA molecules) may be altered and combined to create genetic variation within a species (e.g., recombination of genetic material; mutations; errors in copying genetic material during cell division)

Life Sciences Standard 7 Level IV (Grade 9-12):

2. Knows how organisms are classified into a hierarchy of groups and subgroups based on similarities that reflect their evolutionary relationships (e.g., shared derived characteristics inherited from a common ancestor; degree of kinship estimated from the similarity of DNA sequences)

Life Sciences Standard 12 Level IV (Grades 9-12):

3. Uses technology (e.g., hand tools, measuring instruments, calculators, computers) and mathematics (e.g., measurement, formulas, charts, graphs) to perform accurate scientific investigations and communications

 

How to Sequence Your Students' mtDNA To take advantage of the Dolan DNA Learning Center's DNA sequencing service, participants must first clone a small segment of their mitochondrial genome by Polymerase Chain Reaction (PCR). Two commercial mitochondrial PCR kits are available from Carolina Biological Supply Company: Catalog # 21-1236 DNA Extraction and Amplification Kit includes Chelex, proteinase K, Ready-to-Go Beads (in 0.5 ml tubes), primer/loading dye mix, and DNA markers. Catalog # 21-1237 or 21-1238 DNA Extraction, Amplification, and Electrophoresis Kit includes all of the above, plus agarose, electrophoresis buffer, and either ethidium (1237) bromide stain or CarolinaBLU stain (1238). If you do not want to purchase kits, you can prepare and order the reagents yourself following the 'Recipes' section of the Genetic Origins Web site. Click on the word 'Recipes' highlighted in blue at the top of the Web page. NOTE: Although automated thermalcyclers are convenient for performing PCR, three water baths can be used for 'hand-held' PCR. The mitochondrial PCR reaction requires approximately two hours to perform. Additionally, gel electrophoresis is useful to determine if the mitochondrial DNA fragment has been successfully amplified. Complete shipping instructions along with a registration form can be found here. Click on the blue highlighted 'Sequencing Service' tab in the upper right of the Web page for full directions and the registration form. When completed, number and ship your amplified mitochondrial DNA fragments to: Michael O'Brien Dolan DNA Learning Center Cold Spring Harbor Laboratory 1 Bungtown Road Cold Spring Harbor, NY 11724 The sequencing service takes approximately 10 working days before results are posted on the Sequence Server. You will receive an e-mail notifying you when the sequences are posted.

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