DNA Sequencing
Ethics

DNA Sequencing Module

Working in groups of four, students sequence a fragment of human DNA that has been amplified via Polymerase Chain Reaction (PCR). Separation of DNA fragments is carried out on 50 cm long denaturing gels, and the DNA bands are detected a colorimetric method. The students then interpret their sequencing data.

Our Seattle-based sequencing project is run as an inter-classroom collaboration in which we are shotgun sequencing an expressed region of the human genome. The DNA fragment under study was amplified and the double-stranded DNA templates were purified (done at the UW lab). Each high school classroom sequences at least two different fragments, and after the students have analyzed their data, they enter it into our directory.

Introduction to the Experiment

Prior to starting this lab, the teacher leads the students in a review of the concepts and techniques of DNA sequencing and interpreting a sequencing gel. A modeling exercise using pop-it beads is presented in the introduction to this experiment. Students can also practice interpreting sequencing data using autoradiographs of sequencing gels.

Experimental Day I: DNA Sequencing Reactions

Students carry out standard dideoxy sequencing reactions, using a double-stranded DNA template and a biotin-tagged primer. This can be done in a 55 minute class period.

Experimental Day II: Denaturing Gel Electrophoresis

We use the Bio-Rad Sequi-Gen II sequencing cell because it has several safety features that recommend it for classroom use. This part of the experiment takes about 4 hours, so much of the process needs to be done outside of the class period. The gel apparatus is set up about 1 hour before class time, and the gel is allowed to pre-run in that time. One 6% polyacrylamide-urea gel (38x50 cm) is used for a class of 32 students. During the class period, the students heat-denature their DNA samples, flush the gel wells, load their samples and start electrophoresis. The gels are typically run for about 2.5 hours, although the running times can be varied so that different portions of one DNA tract can be analyzed. At the end of the run, the DNA is transferred to the nylon membrane, which is then irradiated with UV light to covalently crosslink the DNA to the membrane. A narrow strip of membrane is used for each set of four sequencing reactions, so that each student group will have its own membrane to detect and analyze.

Experimental Day III: DNA Detection

Each student group washes its membrane strip, using the technique described for DNA Synthesis. The membranes are allowed to develop for about 16 hours in color substrate in order to get a good signal.

Analysis of Sequencing Data

Each lab group interprets its sequencing gel. These can be photocopied so that each student has a copy. The students analyze their data separately, and then compare their results with those of their labmates and other groups that have sequenced the same fragment. Data assembly is carried out using the Sequencher software program, which is made available to each class on a laptop computer or as a download from the web site. Students can also search the databases via Blast for homologies with other sequences in the DNA databases.

NOTE: Two recently released products could be used for this application. First, Amersham Life Sciences produces a DNA sequencing kit that uses a biotin-tagged primer and contains all the detection components, including nylon membranes. Secondly, Stratagene now sells pre-poured sequencing gels. For a small-scale project, these products may be cost effective.

Ethics Module

Like the Human Genome Project, our program includes an ELSI component that explores many of the complex issues surrounding DNA research. We have focused on the potential of genetic testing because it raises difficult and controversial questions. Our goal is to lead the students beyond a general discussion of the ethical and social issues involved in genetic testing to the point where they can make thoughtful decisions. We accomplish this by providing the students with a thorough scientific background on our model disorder (Huntington's disease), involving them personally through role playing in a scenario about a family that carries the Huntington's gene, and then providing decision making models to enable them to decide whether, as one of the characters in the scenario, they would be tested for Huntington's disease.

Introduction

At the beginning of the ethics unit the students are presented with information about the genetics of Huntington's disease (HD) and its clinical course. Background materials are available for both teachers and students. The students are then presented with a hypothetical case and asked to draw the family's pedigree, using a standard medical genetics format for pedigree construction. Finally, students assume the role of one of the characters in the case and consider whether they would want to be "tested" for the HD gene mutation. They will have an opportunity at the end of the unit to receive mock test results for the character's role they were assuming.

Ethics and Ethical Decisions

The second component of the unit involves a general discussion of the ethical issues that are manifested in the case. Each of five characters in the case is examined to clarify the ethical and personal conflicts that are associated with decisions surrounding HD predictive testing. Then students are introduced to a model for ethical decision making that they will use when considering whether, as a character in the case, they would want to undergo testing. Once students are familiar with the relevant issues and the ethical decision making model, they are asked to use the model to help them determine whether their character should undergo testing.

Testing Decision and Receiving Test Results

In preparation for the third component, students are given a series of discussion questions and an example of an actual informed consent document. They decide whether they want to receive the results of their character's genetic test, justifying their decisions using the principles or values discussed in the decision making process. Students are given a brief overview of informed consent. If they decide to receive their results, they are asked to sign their informed consent forms and are given copies of the mock lab report. Test results are explained to the students and any further questions are answered.

Classroom Strategies

These three modules are routinely presented in one to five classes by each teacher. The venue varies at each school, depending on the number of classrooms and the length of the class periods (usually 55 to 110 minutes). Typically, each module takes about one week to complete. Days I and III of the two experimental modules (synthesizing DNA fragments and detection) can be completed in 55 minutes, although a longer class time is preferable. Day II of the experiments (electrophoresis and transfer of the DNA to nylon membrane) requires approximately 75 minutes for synthesis and 4 hours for sequencing. In 55 minute classes, students are usually able to complete electrophoresis of the mini gels used in the synthesis experiment and can set up the DNA transfer, but then the teacher needs to stop the transfer after the period has ended. Routinely, the large gels used for sequencing are pre-run before the students come to class, students load their samples during the class period and then the teacher stops electrophoresis and carries out the transfer 2 1/2 hours later. The ethics unit is very adaptable and, depending on the length of the classes, can be presented in three to five periods.