CASE TEACHING NOTES
for
"Niños Desaparecidos"

by
Katayoun Chamany
Science, Technology, and Society Program
Eugene Lang College
New School University


INTRODUCTION

This case is based on the actual identification of 50 children who were displaced during Argentina's Dirty War of the 1970s, subjected to DNA and protein analysis, and subsequently reunited with their biological families (4,5,7). The case demonstrates the relationship between society and science and the influences that each has on the other. In addition, the case serves to address the issues of genes in populations, ethnic diversity, statistical significance, bias in interpretation, and gene databanks. Students will learn that the identification of individuals using molecular markers can be a useful tool in certain situations but that each type of analysis has its limitations. With the completion of the Human Genome Project and the Human Diversity Project, an understanding of these concepts is necessary for students to make informed decisions on an individual and societal level. At the completion of the case, students can decide for themselves whether these tests should be used and in what context.

This case study is fiction. The characters (excluding Mary-Claire King) are loosely based on actual people and are not meant in any way to parody any real individuals. Any errors should be attributed to the author, Katayoun Chamany, and not the original investigators.

I have specifically chosen the material described below in an effort to reach underrepresented minority students who often fail to see the connection of science and its applications to their world (much of the resource material is in Spanish as well as English). This case also attempts to address the misconception that science is conducted and shaped only by men. All of the instrumental players in this case are women.

This case can be conducted in a variety of ways depending on the students' academic backgrounds, cultural backgrounds, class size, course objectives, and format. The case uses video- and Internet-accessible images and tutorials in an effort to accommodate all learning styles. Optional genotype testing laboratory activities can be used with the case as well (see Laboratory Activity References).

The case has been used twice, and in both situations the students commented that the multimedia was instrumental for their comprehension of the material and allowed the case to come to life (see Video References). I present two versions for teaching this case here, but the case can be integrated into a more traditional science course in the form of a directed case study or research paper. Regardless of the format, the blocks of analysis will provide the instructor with the necessary background information to be the discussion leader. I am also including a question outline to help guide general discussions. Many of the references are available via the World Wide Web, and are therefore easily accessible to both students and faculty. Of note are the web sites that present information in Spanish. This is particularly useful in reaching Latino/Latina students.

Version One:
This format tackles the issues surrounding the accuracy of genetic identification tests and the construction of genetic databanks. This version can be used easily in a science-based course for majors and takes the form of an interrupted case with optional lab experimentation. For non-majors, the case should be presented in the second half of a course that covers the basics of genetics to ensure that students benefit from this exercise. I have used this format in a course that I teach for non-science majors (the second of a two-semester course at Eugene Lang College). To prepare students in a first-year inquiry course, two to four additional class sessions covering the basics have sufficed in my experience; however, the level of discussion will be less technical and scientific.

Version Two:
This role-playing format tackles the issues surrounding the accuracy of genetic identification tests and the construction of genetic databanks, but with less technical material overall. Students will have the opportunity to explore scientific and non-scientific perspectives in depth through role-playing. This format would work well in any type of course, especially a Science and Society course or first-year inquiry-based course; however, every student will not be responsible for the science of the subject, so content varies from student to student depending on the role that each student plays. The students with strong scientific background would be assigned the roles that require the most background in genetics and/or math.

VERSION ONE

Preparation and Background:
This format for teaching the case can be used in a small (<40) class. The course could be one in the biological sciences or on science, technology and society, or the second half of a first-year inquiry course. Depending on the placement of the case study in the course, the instructor should use discretion when covering the scientific and technical material. It is planned for four 50-minute periods. You will need a VCR/TV, two videos (see Video References Section), and a computer/LCD projection unit with Internet access.

Case Objectives:

  • To teach students to ask good questions.
  • To provide an understanding of genetic diversity.
  • To provide an understanding of the molecular biology techniques needed to conduct the analysis.
  • To evaluate the applications and implications of basic genetic research in society.
  • To consider the social/moral implications of the potential risks involved.
  • To demand a critique of valid conclusions: method of interpretation and acceptable matches.

CLASSROOM MANAGEMENT

Interrupted Case (information will be provided piecemeal)

Session One: Introduction to case and soliciting student questions (TV/VCR)

  1. Show a 15-minute video clip: 60 Minutes: The Dirty War. April 23, 2000.
  2. Ask students: "After viewing this clip, what questions come to mind?"
  3. Spend five minutes gathering student questions. If they are reluctant to pose a question, ask them what they found most surprising or interesting about the video clip; this often stimulates questions from other students. Alternatively, you may pose a question or two, but I think this works best when the students produce their own line of questioning. Sample questions that may elicit more specific questions include: "Are you curious as to how the children were reunited with their biological families?" "Do you wonder which methods and strategies were employed to convict the former fascist leaders?"
  4. Record answers on the board with space to place additional information under each statement.
  5. Distribute the CASE study and allow students five minutes to read the case (do not include the BACKGROUND).
  6. Allow students to assume Isabel's role in the case study and to pose questions from her point of view.
  7. Use five minutes to record their questions on the board.
  8. Distribute the BACKGROUND information and allow students five minutes to read the material.
  9. Ask students to assume Isabel's adoptive mother's role and to pose questions that will serve to clarify the material.
  10. Use 10 minutes to record their questions on the board.
  11. Inform students that they are to select two questions (one social and one scientific) from the board. They are to research the questions outside of class and return the following week with answers (you may choose to have the students complete this assignment in the form of an essay with a bibliography to be turned in during the next class period). I would assign reading to accompany this assignment and the choice would be dependent on the background of the students. For non-majors I would assign the Omni interview of Mary-Claire King, the chapter on DNA fingerprinting from The Code of Codes book, and the Riley web site (5, 10,12). For science majors you could add the seminal article by Jeffreys in Nature and Chapter 10 in Essential Cell Biology by Alberts et al.(10,15).

Session Two: Student information exchange

  1. Have students turn to their neighbor and share their findings for the first 20 minutes of class (10 minutes per student). This exercise forces the students to truly understand their research and to accurately explain it to a peer. The peer must then ask questions and for points of clarification in order to adequately present the new material to the rest of the class. While this is going on, the instructor should identify 6-7 student questions that are different from one another and would be appropriate to share with the rest of the class.
  2. Ask 6-7 students to present two key points from the findings of their peer to the class (five minutes per student).

Session Three: Review of basic genetics, techniques, and applications (Computer/LCD/Internet)

During the class session, use the web sites in the reference section of this case in class to expand on the student findings from the previous session (12,14,17,18,19, 20). During this time be sure to cover the technical aspects of molecular identification techniques. The University of Arizona Human Biology Tutorial web site is very good and is available in Spanish (17). I usually begin the session by covering information that is relatively familiar to most people. Therefore, I begin with a discussion of blood typing and then move on to the more accurate HLA (Human Leukocyte Antigen) typing (9). I then ask the students to identify the limitations of these analyses. Their answers (variance is limited to structure/function relationships) serve as a segue for me to introduce the idea of using genetic sequences to identify individuals rather than proteins. I explain the premise, tools, and method of RFLP (Restriction Fragment Length Polymorphism), and then take the students through VNTR (Variable Nucleotide Tandem Repeat) analysis. Throughout this discussion I emphasize that establishing a level of variance is key to all identification techniques and that the degree of variation in proteins (blood type) is much less than that of VNTR sequences. I wrap up by illustrating that the degree of variance will dictate the level of accuracy, while the methods of the technique will address the level of specificity.

Session Four: TV/VCR

Show a 10-minute video clip from the video "Genetics and Human Rights: Identifying the Children of the Disappeared." This is a video recording of a segment of the Human Genome Project Conference that took place at Stanford University, Palo Alto, California, in 1989 and is available through interlibrary loan to members of the Stanford Library consortium (7). This video will allow the students to see the story, the science, and the personality of the scientist behind this project. There is a section of the video that contains family pedigrees that demonstrates the necessity of classical genetics for molecular genetic analysis. It also demonstrates the need for mitochondrial VNTR analysis. Lastly, the video, through this case study, illustrates that this identification project depends heavily on information from the Human Genome Project, and more specifically the Human Diversity Project to increase the accuracy of the matches. If you are unable to use this video or if you prefer to use a documentary in Spanish, you may choose to show clips from the other videos listed in the Video References section at the end of the notes. "Niños Desaparecidos" and "For These Eyes" are both subtitled, but do not include any science. The Scheck video contains the science but does not include the story of missing children (13). Have the students get into small groups for 20 minutes to discuss the scenario below to arrive at a consensus answer for the dilemma questions for either of the following situations. Each situation deals with issues of genetic databanks, but each approaches the problem from a different perspective (voluntary vs. mandatory samples). End the class session by having members of each group share the consensus answers with the rest of the class.

Situation 1: Facts

  1. You are an adult living in Argentina and have been approached by the Abuelas.
  2. They inform you that you may not be who you think you are.
  3. They ask you to submit a blood sample for genetic identification.

Situation 1: Dilemma Questions—answer these questions and provide your reasoning.

  1. Will you agree to be tested?
  2. Should you be forced to be tested—why and by whom?
  3. If you are forced, who should pay for your test?
  4. Should this information once gathered be destroyed immediately or maintained in the databank?

Situation 2: Facts

The VNTR testing results of Isabel and Claudia Logares have been completed and the results indicate that they are not related. You may choose to show real data or use data culled from the University of Arizona Biology project web site (14). Alternatively, you may have students conduct a simulation experiment during a lab session that demonstrates this. Carolina, Edvotek, and Bio-Rad Explorer all sell kits for these activities (see Laboratory Activities References section).

Situation 2: Dilemma Questions--answer and provide reasoning

  1. How reliable is this result? (students should explain that exclusion is easier to prove than inclusion)
  2. What is your next course of action?
  3. If you were Isabel would you enter your genetic information into the Argentine genetic databank in hopes of a future match?

VERSION TWO

Preparation and Background:
This format can be used in a smaller-sized (<25) class. The course could be one in the biological sciences or on science, technology and society, or a first-year inquiry course. Depending on the placement of the case study in the course, the instructor should use discretion when covering the scientific and technical material. Placing the case towards the end of the course will offer the most benefit to the students, as it requires them to assemble information from many disciplines. It is planned for four or five 50-minute periods. You will need a VCR/TV, two videos (see Video References section), and students should have access and time for library and Internet researching outside of class.

Case Objectives:

  • To teach students to ask good questions.
  • To provide an understanding of genetic diversity.
  • To evaluate the applications and implications of basic genetic research in society.
  • To consider the social/moral implications of the potential risks involved.
  • To demand a critique of valid conclusions: method of interpretation and acceptable matches.
  • To promote the art of collaboration.

INSTRUCTIONS FOR STUDENTS

I. Abbreviated Format for Situational Study

  • We will use this case as a situational study in which you will play the role of an individual involved in this scenario. Roles appear at the end of this page. The case, the background information, the roles, and references will be assigned and distributed during the FIRST SESSION.
  • Outside of class, each of you will use additional resources to develop a character profile appropriate to your role and write a statement that if read aloud would be 3-5 minutes in length. This statement should portray the unique perspective and expertise of your character and include a list of references (see THINGS TO CONSIDER WHILE DEVELOPING YOUR ROLE PROFILE)
  • During the SECOND SESSION, you will bring two copies of your statement to class. One copy will be turned into the instructor and the other will be used in small group work. All students who are assigned the same role will share their statements with one another and work in small groups to develop a composite profile and statement that reveals the types of questions and concerns that the character would find important.
  • During the THIRD SESSION, a member from each role group will read the composite statement that should be four minutes in length. During the presentations, students should record key questions and concerns that appear in the statement. After each statement, students from other role groups will have five minutes to pose questions to the presenting group and these questions and answers should be recorded as well.
  • During the FOURTH SESSION, each group will review the information that was gained during the THIRD SESSION and develop an action plan for Isabel that reflects the interests and concerns of all that are involved in the scenario. Each action plan should be 5 minutes in length if read aloud. All groups will share their plan with the class (see THINGS TO CONSIDER WHILE DEVELOPING YOUR ACTION PLAN).

II. Things to Consider While Developing Your Role Profile

  1. What messages will your character bring to the situation and why?
  2. What kind of information will your character try to gain in this situation?
  3. What kinds of people would your character be most likely to choose as allies?
  4. What will your character present as precedence to back their viewpoint?
  5. What challenges may confront your character in this situation and how will your character defend his or her perspective against an opposing view?

III. Things to Consider While Developing Your Action Plan

  1. What actions should be taken?
  2. What actions would you be less inclined to support?
  3. How will you proceed—what should be done first, second, third?
  4. What are some of the obstacles or challenges that you may encounter while executing your plan and how will you deal with them?
  5. Who is most likely to benefit from your plan and who is most likely to be harmed by your plan?

IV. Evaluation

  1. Is your profile statement clear and informative?
  2. Does your statement reflect your character's personality, concerns, and goals?
  3. Is your action plan realistic?
  4. Does your plan take into account the information that was provided from the other groups and still stay true to your character's perspective?
  5. Did you prioritize your activities?
  6. Does the plan of action provide an appropriate means to overcome the obstacles that may arise during execution?
  7. During discussions did you ask questions of others? Did you challenge those views that contradict your own? Were you adequately prepared for questions from opponents?
  8. Did you respect the members of the class?

INSTRUCTIONS FOR TEACHERS

Before the first class session:

  • Read through the articles, review the web sites, and the student roles carefully.
  • Based on your understanding of class personalities and expertise, assign each student a particular role.
  • Keep a record of these assignments.

In the FIRST CLASS SESSION: Introduce case and assign roles

  • Show the 15-minute video clip: 60 Minutes: The Dirty War. April 23, 2000 (see Video References section).
  • Distribute the case, background, references, and student instructions to all students.
  • Explain the role-playing activity and be sure to mention that there will be multiple people playing one role (there are five roles, so in a class of 20 there would be four students sharing the same role).
  • Make certain that they understand the task: they are to develop a profile for the role they have been assigned through library and Internet research outside of class.
  • Ask if there are any questions, as this activity will be new to most of them.
  • Assign the roles to the students.

In the SECOND SESSION: Student information exchange, small group work

  • Collect the statements.
  • Allow students sharing the same role to work in small groups to develop a composite role profile. Alternatively, you can have each student read his or her statement aloud to the class and only allow questions to be posed from those students who share the same role.

In the THIRD SESSION: Presentation of composite roles, Q & A

  • One member from each ROLE group will read the composite role developed by that group.
  • While that student is reading, other students will record the key statements and questions that this character makes during the reading of the statement.
  • After the reading, the members of the other role groups can pose questions to this group.

In the FOURTH SESSION: Develop and share composite action plans

  • The class will assemble into their ROLE groups.
  • Each group will review the information that was gained during the THIRD SESSION.
  • Each group will develop an action plan for Isabel that reflects the interests and concerns of all that are involved in the scenario.
  • Each action plan should be five minutes in length if read aloud. All groups will share their plan with the class and notes should be recorded.

To summarize:

  • Each ROLE group will be made up of 4 members.
  • Member 1 is responsible for reading the composite statement.
  • Member 2 is responsible for recording key statements and questions of another ROLE group's statement.
  • Member 3 is responsible for answering any questions that are posed by the other ROLE groups.
  • Member 4 is responsible for reading the composite action plan.

You may wrap up with some general questions:

  • Whose voice was not heard in this scenario and why?
  • Do you think that science can improve the quality of life?
  • Do you agree with DNA databanks and if so, in what context?

If time allows, you may choose to show a 10-minute video clip from the video "Genetics and Human Rights: Identifying the Children of the Disappeared." This is a video recording of a segment of the Human Genome Project Conference that took place at Stanford University, Palo Alto, California, in 1989 and is available through interlibrary loan to members of the Stanford Library consortium (7). This video will allow the students to see the story, the science, and the personality of the scientist behind this project. There is a section of the video that contains family pedigrees that demonstrates the necessity of classical genetics for molecular genetic analysis. It also demonstrates the need for mitochondrial VNTR analysis. Lastly, the video, through this case study, illustrates that this identification project depends heavily on information from the Human Genome Project, and more specifically the Human Diversity Project to increase the accuracy of the matches. If you are unable to use this video or if you prefer to use a documentary in Spanish, you may choose to show clips from the other videos listed in the Video References section at the end of these notes. "Niños Desaparecidos" and "For These Eyes" are both subtitled but do not include any science. The Scheck video contains the science but does not include the story of missing children (13).

ROLE PROFILES

I suggest that you only give the title of the role to the students. The comments made here are for the instructor's use only. If your students need more guidance, I recommend that you design a short list of questions for each role based on the content that has been covered in your course so far.

ROLE 1: Genetics Counselor: This profile should illustrate the importance of classical and molecular genetic analysis and the training required to become a genetic counselor. Questions may include those that revolve around obtaining informed consent. The profile should also address how a geneticist calculates the frequency of a particular genotypic match. The challenges should include locating enough living relatives, obtaining a large enough number of molecular markers, and considering how genes behave in populations and how that may affect your calculation.

ROLE 2: Lawyer for the Abuelas de Plaza de Mayo: This profile should depict the expertise of the lawyer. This character should provide precedence for Isabel's case by presenting cases in which DNA evidence was used and explaining why these cases were either won or lost. This role also demands that the lawyer explain why a negative match is more reliable than a positive match.

ROLE 3: Commercial Gene Testing Laboratory Representative: This profile should include a description of the services provided by the company and the parameters used in making positive matches. Other members in the situation on at least two accounts will challenge this company: incidence of identifying false positives and courts that do not admit DNA evidence.

ROLE 4: A Member of the Abuelas de Plaza de Mayo. This profile should include the entire history of the Argentine War and compare this war to others in which children were displaced. Statements may include a more broad range of goals for the abuelas—to educate others about their plight and to help other women around the world find their lost children. Challenges may include lack of cooperation by government officials and loopholes in the law that prevent the abuelas from prosecuting the fascist parties.

ROLE 5: Member of the Council for Responsible Geneticists or Physicians for Human Rights: Again, this profile should include a mission statement for the organization and cite some specific examples of their involvement in human rights issues. Questions may include the role of government, the United Nations, and the victims during and after war crimes are committed.

BLOCKS OF ANALYSIS

ARGENTINA'S DIRTY WAR

During the 1960s and 70s, Argentina was a land of extreme social and political unrest. This climate was the result of a series of events that led the Argentine right and the military junta to take quick action to eliminate the perceived subversives that were gaining high profile during this time. Democratic leader Salvador Allende had just been recently elected into office in Chile, Cuba's youth was embracing communist regime, and the Catholic Church was losing its hold in Argentina (6). Thus, the Argentine right felt that the ushering in of liberal values would quickly lead to a communist structure that would destroy the conservative rule. Their primary targets were the educated, and this included academics, students, and journalists (5,6,7). In 1976, the military took over and employed a national security doctrine that provided a procès de mise en acceptablité—that is, a process of "making acceptable what is not considered normal, decent, or adequate in the first place" (6,21). Within days, 3,000 university professors were dismissed from their posts; most were arrested shortly thereafter. Amnesty International reported that 200 intellectuals and students disappeared and were never seen again (4,6). Between 1976 and 1983, the military continued to rule under a policy called the "Process of National Reorganization," or the "Proceso," which allowed the junta to attack, kidnap, and torture guerillas and civilians (6). The Argentine Truth Commission estimates that 9,000 people were killed by the junta, but this number falls short of those put forth by human rights groups such as Amnesty International, the Lawyers Committee for International Human Rights, and the Buenos Aires Center for Legal and Social Studies. These groups corroborate on the following statistics: a death toll of 15,000; tortured but not killed at around 30,000; and those that were exiled at 500,000 (2,5,6,21).

After the war, President Alfonsin released the report Nunca Mas, put forth by the National Commission on the Disappeared (CONADEP). The information gathered in this report suggests a more far reaching devastation than was originally proposed (4). To add insult to injury, in the years that followed the Dirty War, many steps were taken by the democratic government to allow military officials to escape punishment by law. The first of these was to honor a self-imposed "Law of National Pacification," granting immunity from prosecution to suspected terrorists and to every member of the armed forces for crimes committed between May 25, 1973, and June 17, 1982 (6). However, in 1983, the Argentine Congress repealed the law and prosecution of military leaders began under the supervision of President Alfonsin. The Argentine people felt a sense of justice when five former commanders were convicted and given sentences ranging from four and a half years to life imprisonment (6). The floodgates had opened and the Argentine court system was overwhelmed with cases. Unfortunately, the situation made Alfonsin more amenable to pressures being placed on him by military officers and in 1987 he passed the Law of Due Obedience, which did not allow military officials to be charged with crimes (6,22,23). In 1990, President Carlos Menem took these actions a step further and pardoned about 280 members of the military who still faced trial for human rights abuses. In 1998, the Argentine Congress passed a measure to cancel the Law of Due Obedience and the Final Point (Punto Final) and today some of the military leaders are on trial (16,23).

IDENTIFICATION OF INDIVIDUALS USING MOLECULAR MARKERS

The history of using molecular markers to identify individuals illustrates a progressive trend toward methods that are more sensitive and offer more accurate results. Originally, the choice of proteins used in paternity and forensic tests were the blood type proteins, but this only allowed for the detection of four different phenotypes (A, B, AB, O) (17). On a genotypic level there were more permutations, but this was still limited to only six distinct genotypes (A-, AA, AB, B-, BB, --). The homozygote (AA) could be distinguished from the heterozygote (A-) only by using quantitative RT-PCR (reverse transcriptase polymerase chain reaction, a technique that measures the level of expressed gene products at the mRNA level).

The choice of proteins used in these tests is the HLA, Human Leucocyte Antigens. These proteins are variable in populations and allow the human body to recognize self from non-self. Sometimes these proteins are called the MHC, or Major Histocompatibility Complex. A large cluster of genes located on chromosome 6 codes for the subunits of the HLA proteins (8,9). Each person receives genetic information for each protein from each of his/her parents. Within a population, however, there are multiple versions (alleles) for each gene. Therefore, this locus is considered highly polymorphic. Typically, polymorphic genes encoding proteins are represented by 2-6 alleles. One subunit of one of the HLA proteins (DQalpha), involves the detection of six alleles that define 21 genotypes (8). The HLA proteins and the genes that code for them help researchers identify people based on the detection of specific variants. In the case of the missing children, researchers based their studies on the simple fact that related individuals should share more alleles than non-related individuals.

However, as investigations proceeded over the years it became clear that there was still an unacceptable probability of finding a match between two unrelated individuals by chance. In Caucasian populations, the chance that any two individuals would share the same DQalpha genotype is about 7% (8). The degree of variability at these loci was not high enough, or put another way, the chance that two individuals chosen at random would share the same profile for these loci was too high.

Analyzing genetic sequences that are highly variable in the population solved this problem. These sequences are scattered in the human genome and if compared among individuals would reveal differences in length due to a variable number of tandem repeats (VNTR) (10,11,12,13,14,15). A tandem repeat is a short sequence of DNA that is repeated in a head-to-tail fashion at a specific chromosomal locus. One example of a VNTR in humans is a 17 bp sequence of DNA that may be repeated between 70 and 450 times in any one person's genome. When considering the whole population then, the total number of base pairs at this locus could vary from 1190 to 7650 base pairs. There are many of these VNTR loci in the genome, each in a distinct location. For each person, one copy of this VNTR locus came from each parent and the length of these can be different.

Each VNTR sequence is located in a specific region of the genome and this location is maintained from individual to individual. Only the length of each VNTR will vary among individuals, not the location. Therefore, you can compare the length of the VNTR sequences amongst individuals and establish whether individuals are related.

To conduct this analysis, researchers enrich (make many copies of) a specific VNTR sequence from one individual's genomic DNA using the polymerase chain reaction (PCR) and single-stranded DNA primers that are complementary to the flanking DNA of the VNTR locus (15,20,18). The flanking sequences are highly conserved in a population and appear at either end of the VNTR locus. Amplification of the VNTR sequences allows researchers to obtain enough VNTR DNA to make size comparisons. The technique is done in vitro and involves separating the DNA into its two complementary strands. Single-stranded DNA primers anneal to the conserved sequences at either end of the VNTR region. Then a thermostable DNA polymerase recognizes the primers and copies each strand of the VNTR sequence between the primers. The process is repeated many times, leading to an exponential increase in copy number of the VNTR sequence.

Once the region has been amplified, comparisons can be made with VNTR DNA that has been amplified from another individual using the same single-stranded DNA primers. The PCR products from both samples will be subjected to gel electrophoresis and the products separated based on size. DNA is loaded into a gel and a current is applied. The DNA migrates through the agarose gel towards a positive pole since it possesses a negatively-charged phosphate backbone (15,20). The gel acts like a sieve. Fragments that are longer in length are retarded in the gel matrix, while shorter fragments, able to squeeze their way through the matrix, move more quickly though the gel. After a certain amount of time has passed the current is removed, the DNA in the gel is stained using ethidium bromide, and the PCR products are viewed under UV light. The longer fragments remain closer to the starting point than the shorter fragments that will have migrated further away from the starting point. When comparing samples from different individuals, one should observe that the PCR products vary in length. Unrelated individuals should have very few VNTR sequences in common and therefore should not share many PCR products of the same length. On the other hand, related family members should share some of the same VNTR sequences with one another and, therefore, the analysis should display some VNTR sequences of the same length. Many of the steps in this protocol have been eliminated due to the development of more advanced technologies that capitalize on the information from the Human Diversity Project. Many companies now use DNA identification dot blots (12).

Alternatively, DNA can be collected from individuals and subjected to restriction fragment length polymorphism (RFLP) analysis (7,13,15,20). In this assay, genomic DNA is digested with restriction enzymes that are known to flank the VNTR regions. The entire genome will be fragmented and these fragments are separated based on size using gel electrophoresis. To specifically detect and analyze the migration of the VNTR sequence to be tested, one must then conduct Southern Analysis (15,20,14). This analysis requires that the DNA be immobilized maintaining the pattern obtained by gel electrophoresis. Thus, the DNA in the gel is denatured, making it single stranded, and then transferred to a positively charged nylon membrane. The membrane with the attached DNA is mixed with a fluorescent (or radioactively labeled) single stranded DNA that is complementary to the VNTR sequence of interest. This DNA is called the probe. Since both the target and the probe are single stranded and complementary, they will hybridize and you will be able to detect the signal via fluorescence or autoradiography and be able to make size comparisons.

Both PCR and Southern Analysis take advantage of the double-stranded nature of DNA. These techniques melt the sample DNA helix, producing single stranded DNA molecules that will hybridize to complementary sequences. In the case of PCR, the complementary sequences are single stranded DNA primers and in the case of the Southern, the complementary DNA sequence is a single stranded-labeled DNA probe. The VNTR sequence extracted from the blood sample of an individual serves as the target, while the primer and the probe serve to identify the VNTR sequence.

Regardless of the method used, in a typical identification test, 4-6 different VNTR loci will be analyzed simultaneously to assure that a match does not happen by chance and is statistically significant (11). This type of analysis narrows the chance of two individuals having the same length VNTR pattern (DNA fingerprint) down to about a million to one. There is controversy about how this is determined. Not surprisingly, the product rule is applied, which assumes that the probability of inheriting one allele of a VNTR locus is independent of inheriting any allele at another VNTR locus. Given the preliminary study of genes in populations, research suggests that this is not a correct assumption; that in fact, some alleles are associated with particular ethnic populations and therefore the loci are not truly independent of one another.

In the case of the Abuelas, the number of living relatives who could provide DNA samples was low due to the assassinations of the parents. Therefore, HLA typing was not accurate, and VNTR testing of the nuclear genome did not allow for enough points of reference (not enough living relatives). Realizing that the maternal lineage was still intact (living grandmother), King used the highly variable sequences of the mitochondrial genome to trace the children back to their biological families. Since mitochondria are maternally inherited, every child could be traced back to a grandmother (24). Another advantage to using the mitochondrial genome is that it mutates at a faster rate than the nuclear genome, making it very unlikely to find two grandmothers with similar mitochondrial DNA and therefore much less chance for false positive matches (24).

DNA EVIDENCE IN COURTS

The use of DNA evidence in court cases proves to be a rather sketchy matter in that the decision to admit such data is based on the Frye rule established in 1923:

…while courts will go a long way in admitting expert testimony deduced from well-recognized scientific principle or discovery, the thing from which the deduction is made must be sufficiently established to have gained general acceptance in the particular field to which it belongs.
Frye v.U.S, 293F. 1030 (1923).

Lander points out that DNA analysis has been widely accepted in medical applications, but that these conditions vary a great deal from the conditions used when the samples are obtained from crime investigations (11,18). In the latter scenario, the DNA extraction method may vary for each of the samples, thereby causing band shifts, excessive restriction enzyme cutting, or incomplete digestion—all of which would skew the results of such a study. This does not really pertain to the case study here, in that the samples could be obtained by the same methods from both grandmother and child if both are still living. More specific to the Abuelas situation is the following point: If we compare the medical application of establishing paternity or genotype of a child, we usually have only one or two alternative VNTR banding patterns with which to compare (the mother and the potential father's banding patterns). The mother in this situation serves in some ways as an internal control. The child should share some VNTR sequence patterns with her mother. In the case of the Abuelas and the lost grandchildren, the maternal lineage of mitochondrial VNTR sequences dictate that a grandmother and lost grandchild should share the same mitochondrial VNTR banding pattern.

In the mid-1980s, many private gene testing companies sprang up (Cellmark and Lifecodes) and DNA evidence began to enter our court systems, but not without trouble. Lander provides personal testimony that causes one to question the level of blatant human error of these testing companies, the lack of normalization in interpreting results, and the overuse of exclusion tactics by defense lawyers (11,25). The latter point stems from the fact that it is much easier to prove that two samples are not a match than it is to prove that they are. For these very reasons, the U.S. National Academy of Sciences has established a committee to recommend standards for DNA fingerprinting.

GENE DATABANKS

If you choose to cover this topic in detail you may want to purchase the video/transcript of the Scheck talk listed in the Video References section so that you can show this video to the class as well. With the automation of genetic technologies, including DNA sequencing, a number of human genome projects emerged in the late 80s and early 90s and the completed rough draft of the Human Genome Project in April of 2000 stunned the world (26,27). What may come as more of a surprise is the way in which this new technology and information will be used. Nations with homogeneous populations have established their own genome databases. Iceland is breaking ground on this unknown territory and, as if one company holding the holy grail of a nation was not enough, Iceland now has two genome projects: the privately funded DeCode and the government funded Genome Center Foundation (28). Other countries have chosen to construct databases on a need-to-know basis in an effort to curb crime and to close unsolved cases. The UK has established a criminal database that contains the DNA of every criminal and the DNA from the scenes of unsolved crimes, totaling 463,000 samples, and they expect to have cataloged 5 million samples by the end of the year 2000 (13). To date they are solving about 300-500 crimes a week with this relatively new system. The United States has followed suit, and there is legislation in 50 states for forensics DNA databanks, although they are only operational in 36 states (13). The United States National Criminal Database, CODIS, is underway and uses only the most sensitive of DNA tests, the PCR-STR (single tandem repeat), in which 13 different sections of DNA are analyzed (14). The United States also hopes to have 4 million samples from the Armed Services cataloged by 2001 despite the protests of Hawaiian Marines who are concerned about disclosure to other organizations (13). For years, Jewish Centers in New York City have cataloged the DNA of single Jews and offer matchmaking advice based on compatibility of the DNA samples, due to the high rate of Tay-Sachs and other recessive lethal diseases in this population. The databank craze has spilled over to very specialized groups, including IVF clinics, blood banks, and private employers.

GENERAL DISCUSSION QUESTIONS

  1. Before genetic testing was used for paternity tests, blood samples were taken from all potential parents and the child in question and analyzed.

    • What type of analysis was performed?

    • What type of evidence would support that a particular male was the father of a child in question? What type of evidence would indicate that he was not the father of this child? Which of these data is more conclusive?

  2. With regard to using proteins versus DNA for identification:

    • Which is more accurate?

    • Which is more appropriate in Isabel's situation?

    • a) Which DNA sequences or proteins would you use to conduct your analysis?

      b) What type of analysis would you perform?

      c) What types of controls would you include to ensure that your tests were valid?

      d) Draw results that would indicate that Claudia is Isabel's grandmother.

  3. Identification of individuals using biological markers has become commonplace. Describe other situations in which identification has been used and include the individuals and the techniques involved.

GENERAL DISCUSSION ANSWERS

  1. Before genetic testing, blood type or HLA typing was performed. Basically, a child who had a protein type that could not be found in the mother or the potential father would exclude this male from being the father. If the child shared protein types with the potential father, it would support paternity but it would not prove paternity. The lack of a definitive answer lies in the fact that proteins perform specific functions based on their structures and, therefore, little variation occurs within a population. This is especially true for blood type proteins, which only come in two varieties (A or B). The degree of variation is increased with respect to the HLA (MHC) proteins, as these proteins have evolved to respond to changing environments and are therefore polymorphic.

  2. Since some sequences of DNA do not code for protein, these sequences are not constrained by structure/function relationships and therefore are highly variable. Given that in most cases only the grandmothers and children survived the war, mitochondrial VNTR sequence analysis would be the best choice for identification purposes. In conducting this analysis, it is important to include a positive control (purified VNTR sequence as one of the samples) for the efficacy of the technique and reagents, and to eliminate human error. A negative control (no DNA sample) is also included to ensure that none of the samples were contaminated during the procedure. This negative control is extremely important, as PCR is very sensitive and can amplify minute amounts of DNA. If Claudia is Isabel's grandmother they should have the same mitochondrial VNTR pattern. You may want to walk through the results of the Blackett Family DNA test for an illustrated example of this (14).
  3. REFERENCES

    1. Rosenberg, T. February 7, 1992. What did you do in the war, mama? The New York Times Magazine 52.
    2. McHale, Laurie. Putting the puzzle together: in the jigsaw world of human genetics, UW professor Mary-Claire King found a crucial piece that helps solve the mystery of breast cancer. University of Washington Alumni Association web site: September 1996 columns. <http://www.washington.edu/alumni/columns/sept96/king2.html> (Site available as of 01/04/01)
    3. Gibb, T. Missing children haunt Salvador: campaigners are pressing for army units to be brought to justice for their civil war atrocities against the very young. The Guardian, Manchester (UK). Oct. 13, 2000:19.
    4. Argentina's National Commission on Disappeared People. Nunca Mas (Never Again). English Edition. London, Boston: Faber and Faber in association with Index on Censorship: 1986. Also available at <http://www.nuncamas.org>*** (Site under construction as of 03/01/01)
    5. Mary-Claire King (Interview). July 1993. Omni 15(9):68.
    6. Slawner, K. Interpreting victim testimony: survivor discourse and the narration of history. The Vanished Gallery (web site). < http://www.yendor.com/vanished/karenhead.html> (Site available as of 01/04/01)
    7. King, M. Genetics and human rights: identifying the children of the disappeared. Part of the series The human genome project [videorecording] : biological nature and social opportunities. Executive producer John O. Green. Stanford Alumni Association. Stanford University, Palo Alto, California.
    8. Hui, K.M., and J.L. Bidwell, eds. 1993. The handbook of HLA typing technique. Boca Raton, Florida: CRC Press.
    9. Ng, J. et al. Interpretation of DNA-based typing of HLA and correlation with serologic types for bone marrow transplantation: A guide for transplant. BMTinfo (web site), Georgetown University. 1994. <http://www.bmtinfo.org/bmt/topics/htm/type_b.htm#antigens> (Site available as of 01/04/01).
    10. Jeffreys, A.J. et al. Hypervariable 'minisatellite' regions in human DNA. March 7, 1985. Nature 314(6006):67.
    11. Lander, E. DNA fingerpinting: science, law and the ultimate identifier. In The Code of Codes: Scientific and Social Issues in the Human Genome Project. Kelves and Hood, ed. 1992. Cambridge, MA: Harvard University Press, p. 19.
    12. Riley, D.E. DNA testing: an introduction for non-scientists—an illustrated explanation.1998. Scientific Testimony: An Online Journal. <http://www.scientific.org/tutorials/articles/riley/riley.html> (Site available as of 01/04/01)
    13. Scheck, B. Privacy: The impact of DNA databanks. Seminar given at the Jacob Burns Ethics Center, Cardozo Law School, March 2, 1999.
    14. Blackett family DNA activity. University of Arizona: The Biology Project (web site). <http://www.biology.arizona.edu/human_bio/activities/blackett/introduction.html> (Site available as of 01/04/01).
    15. Alberts, B. et al. 1998. Essential Cell Biology. New York: Garland Publishing.
    16. The dirty war. Segment of 60 Minutes, originally aired April 23, 2000. Videorecording. New York: CBS Worldwide Inc.
    17. Blood types tutorial. University of Arizona: The Biology Project (web site). <http://www.biology.arizona.edu/human_bio/problem_sets/blood_types/markers.html> (Site available 01/04/01). ***
    18. DNA forensics problem set 1. University of Arizona: The Biology Project (web site). September 2000. <http://www.biology.arizona.edu/human_bio/problem_sets/DNA_forensics_1/DNA_forensics.html> (Site available as of 01/04/01).
    19. The DNA files: is our fate in our genes? 1998. Soundvision Productions. <http://www.dnafiles.org/about/index.html> (Site available as of 01/04/01).
    20. Access Excellence Graphics Gallery (web site). Genentech. <http://www.accessexcellence.org/AB/GG> (Site available as of 01/04/00).
    21. Perelli, C. From counterrevolutionary warfare to political awakening: the Uruguayan and Argentine armed forces in the 1970s. Fall 1993. Armed Forces & Society 20(1):25.
    22. Yendor of Yonder. The Vanished Gallery (web site). <http://www.yendor.com/vanished/index.html> (Site available as of 01/04/01).***
    23. Yendor of Yonder. Military uprisings. The Vanished Gallery (web site). <http://www.yendor.com/vanished/uprisings.html> (Site available as of 01/04/01).***
    24. Lewis, R. Mitochondria-eclectic organelles. October 1989. Biology Digest 16:202.
    25. Witkin, G. Science takes a stand. July 11, 1994. U.S. News & World Report 117(2):29.
    26. Gosselin, P. Human genome project objects to rival's Science journal deal. The Los Angeles Times [Home Edition]. December 13, 2000:C.1.
    27. Marshall, E. Human genome: storm erupts over terms for publishing Celera's sequence. December 15, 2000. Science 290(5499):2042.
    28. Lone, F. Storm brews over gene bank of Estonian population. November 12, 1999. Science 286(5443):1262.

    ***These sites have articles and translations in Spanish.

    VIDEO REFERENCES

    1. King, M. "Genetics and Human Rights: Identifying the Children of the Disappeared." Part of the series The human genome project [videorecording] : biological nature and social opportunities. Produced and copyrighted by the Stanford Alumni Association in 1991. Executive producer John O. Green. Stanford Alumni Association, 1991. 8 videocassettes (VHS)

      Available only through interlibrary exchange for consortium members (no longer sold): Cecil H. Green Library, Stanford University. Phone (650) 725-6243. Refer to the call number ZVC 4647. <http://www-sul.stanford.edu/depts/ils/ill_plcy.html>

    2. Brava, E. "Niños Desaparecidos." In Spanish with English subtitles. 1 videocassette (25 min). Cinema Guild, New York, NY. 1985.

      Available through interlibrary exchange for consortium members (no longer sold): Cecil H. Green Library, Stanford University. Phone (650) 725-6243. Refer to the call number ZVC 6438. <http://www-sul.stanford.edu/depts/ils/ill_plcy.html>

      Also available from Cinema Guild, 1697 Broadway, Suite 506, NY, NY 10019. Phone (212)-685-6242. This video costs $250 to purchase. It costs $55 for a 3-day rental (+$11 shipping and handling) and the video will be shipped UPS (takes about one week). (As of 01/08/01) <http://www.cinemaguild.com/>

    3. Arijon, G., and V. Martinez. "For These Eyes." In Spanish with English subtitles. 52 minutes. Brooklyn, NY. 1998.

      Available from First Run/Icarus Films, 32 Court Street, 21st floor, Brooklyn, NY 11201. Phone (718) 488-8900. Fax 718-488-8642. This video costs $390 to purchase. It costs $75 for a rental. <http://www.frif.com/new99/forthese.html>

    4. CBS Worldwide Inc. "60 Minutes: The Dirty War." 15 minutes. New York, NY. Originally aired April 23, 2000.

      Available for purchase for $29.95. To order, call 800-848-3256. To order a transcript, call (800) 777-TEXT. <http://cbsnews.com/now/story/0,1597,13504-412,00.shtml>

    5. Scheck, B. "Privacy: The Impact of DNA Databanks." Seminar given at the Jacob Burns Ethics Center, Cardozo Law School, March 2, 1999.

      Available for purchase for $25.00 through the Cardozo School of Law. Phone 212-790-0368.

    6. Carolina Biological Supply Company. "Restriction Enzyme and DNA Video." A 30-minute video explaining and demonstrating the concepts of restriction enzymes and gel electrophoresis can be ordered from this company using catalog #BA-21-1189 ($21.00). Phone 1-800-227-1150. <http://www.carolina.com>

    LABORATORY ACTIVITIES REFERENCES

    1. Carolina Biological Supply Company. "Exploring electrophoresis and forensics." Field tested by teachers at the 1978 NSTA Convention in Las Vegas. This technically simple experiment simulates the use of DNA in forensic investigations. Students cast agarose gels, load predigested DNA, and perform electrophoresis. The banding patterns of the DNA in the gel are used to compare the "DNA fingerprints" of 2 suspects with evidence DNA. Can be ordered from the 2000 edition of the catalog using the Catalog #BA-21-1014 ($180.00/10 workstations) Phone 1-800-227-1150. <http://www.carolina.com> A 30-minute video explaining and demonstrating the concepts of restriction enzymes and gel electrophoresis can also be ordered from this company using catalog #BA-21-1189 ($21.00). <http://www.carolina.com>
    2. Carolina Biological Supply Company. "Human mitochondrial DNA kit AT." Using PCR, students amplify a 460-nucleotide sequence within the control region of the mitochondrial genome. This region contains no genes and has a high mutation rate. While all students in a class will show an amplification of the same size, this is the easiest of all human amplifications to perform. Amplified student samples can be submitted to the DNALC's Sequencing Service, which will generate student DNA sequences and post results on the Internet for a modest fee. These sequences can be used at the online DNA Sequence Analysis facility at the DNALC web site (http://vector.cshl.org/) to explore theories of human evolution and solve cases in forensic biology. Can be ordered from the 2000 edition of the catalog using the Catalog #BA-21-1236 ($129.00/10 workstations) Phone 1-800-227-1150. <http://www.carolina.com>
    3. Carolina Biological Supply Company. "Human VNTR polymorphism kit AT." This kit assays for a VNTR polymorphism that is caused by short repeated copies of a 16bp sequence at the pMCT118 locus. Using PCR and gel electrophoresis, students detect differences in the number of repeated units (longer or shorter alleles). Because the VNTR locus has more than 20 different alleles, a panel of student types shows a variety of genotypes. Can be ordered from the 2000 edition of the catalog using the Catalog #BA-21-1233 ($129.00/10 workstations) Phone 1-800-227-1150. <http://www.carolina.com>
    4. Bio-Rad Explorer. "DNA fingerprinting kit." This kit simulates the DNA testing in a forensic context, but with some modifications can be used in any context. Students use restriction enzymes to cut 5 hypothetical samples of human DNA. One sample represents that of the crime scene and the other four are obtained from possible suspects. The samples are analyzed using gel electrophoresis. Can be ordered from the 2000 edition of the catalog using the Catalog #166-0007-EDU ($69.50/8 workstations) Phone 1-800-424-6723. Free curricula are also available at <http://www.explorer.bio-rad.com>.
    5. Edvotek. "DNA paternity simulation." This kit uses predigested DNA samples to demonstrate DNA paternity testing. Can be ordered from the 2000 edition of the catalog using the Catalog #114-Q ($86/12 workstations) Phone 1-800-338-6835. <http://www.edovotek.com>
    6. Edvotek. "PCR-based VNTR human DNA typing." This kit assays for a VNTR polymorphism that is caused by short repeated copies of a 16bp sequence at the D1S80 locus. Students collect their DNA from cheek cell and using PCR and gel electrophoresis detect differences in the number of repeated units (longer or shorter alleles). Can be ordered from the 2000 edition of the catalog using the Catalog #334 ($145/24 workstations) Phone 1-800-338-6835. <http://www.edovotek.com>

    Acknowledgements:This case study was developed as part of a National Science Foundation-sponsored Case Studies in Science Workshop (NSF Award #9752799) held at the State University of New York at Buffalo on June 7-11, 1999.


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