In the Laboratory
Chromatin Isolation and DNA Sequence Analysis in Large Undergraduate Laboratory Sections
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Ann E. Hagerman Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056;
[email protected] All areas of biochemistry have been impacted by the rapid growth of molecular biology over the past 20 years. Parallel advances have been made in our understanding of the structure and function of DNA and in our ability to manipulate DNA. A variety of tools such as the polymerase chain reaction (PCR), site-directed mutagenesis, cloning, and antisense RNA are widely available. These tools have been used to address questions about protein chemistry (1), metabolic control (2), and enzyme mechanism (3) and have been used in the legal courts (4 ) and in industry (5). Given the impact of molecular biology in research, industry, and daily life (6 ), it is imperative that even introductory biochemistry courses provide a sound background in molecular biology. Current Pedagogy Popular biochemistry textbooks have ably addressed the need for explanation and illustration of the principles and applications of molecular biology. Laboratory instruction in molecular biology at the advanced level has been addressed in lab manuals (7 ) and in the educational literature (8). Many techniques used in molecular biology are relatively routine and are even available as commercialized kits, which are particularly appealing for classroom use (9). Experiments like these have been successfully used at many colleges and universities in advanced biochemistry courses. For example, in small lab classes (10–12 students) molecular biology techniques such as restriction enzyme mapping, Southern blotting, and the PCR can be routinely performed by students. Virtually all these laboratory exercises can be conducted only in a well equipped lab. Each student must have ready access to small equipment such as micropipets, tabletop microfuges, and constant-temperature baths and to major equipment such as UV spectrophotometers, electrophoresis apparatus, and high-speed centrifuges. Specialized equipment including PCR machines or gel-scanning software may be necessary. In some cases, facilities for culturing microbes, including sterilizing hoods, incubators, and autoclaves are essential. In addition to the equipment costs associated with these experiments, a significant budget is required for expendable supplies, including plastic tips, tubes, and petri dishes, reagents, and biochemicals such as restriction enzymes. An additional significant limitation to most molecular biology experiments is the time and technical assistance required for their successful completion. Relatively long lab periods and periods on successive days are essential for many molecular biology experiments. Accidental contamination or sloppy pipetting can cause even commercial kits to fail, and a low student-to-teacher ratio is essential for most of these exercises. Large Laboratory Sections Molecular biology experiments suitable for large-section undergraduate classes have not been published. The supplies 1426
and equipment required for even relatively simple DNA sequencing or PCR experiments make it impossible to implement those techniques in classes like our introductory onesemester biochemistry class, which has an enrollment of 200–250 students. It is essential for these students, who are majoring in subjects such as life sciences, dietetics, or physics, to have at least some exposure to the tools of modern molecular biology. The two-week sequence of laboratory work and computer simulations described here has been very successful in large-section laboratory classes (75 students working with three teaching assistants and one faculty member). The exercises introduce the fundamentals of DNA chemistry and illustrate the relationship between gene and protein. They require minimal equipment and supplies, are technically simple, and provide the students with a fundamental understanding of molecular biology, which can be amplified in later courses.
Chromatin Isolation The first exercise is an experiment in which the students isolate chromatin, the protein–DNA complex comprising eukaryotic chromosomes, from chicken liver obtained at a local grocery store. They then separate the histones, which are lysine- and arginine-rich proteins, from the DNA by ion exchange chromatography using columns prepared in disposable transfer pipets. Fractions collected during the chromatography are qualitatively analyzed for protein using ninhydrin, which reacts with aliphatic amino groups to form a purple chromophore. DNA is qualitatively detected with Bial’s reagent, which yields a blue chromophore with pentoses. All reagents and equipment required for this experiment are routinely available in instructional laboratories. The experiment can be completed in a three-hour lab period. Simulated Gene Analysis The second exercise uses computer simulations as an attractive and realistic alternative to experimental DNA sequencing and gene analysis. The simulation is divided into four parts. First the students learn to read the sequence from Maxam and Gilbert or Sanger reaction mixtures using a simple computer program that simulates the results obtained from random sequences. They then solve a sequence “puzzle” by overlapping restriction fragments generated from a segment of DNA to obtain the partial sequence of a gene. Computer software is used to analyze and translate a DNA sequence that is provided as a computer file. Finally, a “BLAST” search is performed on the amino acid sequence of the protein using the Web site of the National Center for Biotechnology Information. To use this series of simulations, the instructor must ensure that students have access to PCs that can access the World Wide Web. The two software packages, DNA Sequencing (10) and Visual Sequence Editor (11), must be properly licenced and installed. The DNA sequence, which the instructor downloads from the National Center for Biotechnology
Journal of Chemical Education • Vol. 76 No. 10 October 1999 • JChemEd.chem.wisc.edu
In the Laboratory
Information database (as described in the online material that accompanies this article),W must be provided to the students as a file either on the computer or on diskette. The DNA sequence that is to be analyzed must be selected carefully to ensure that the exercise is simple enough for the students to complete. Analysis of the simple bacterial gene bgaC (Accession number D88750), which encodes the β-galactosidase-3 from Bacillus circulans (12), has been successfully used in our class. The simulation can be completed in three hours by students who have had some previous experience with a computer. Acknowledgments Chris Makaroff and Brenda Blacklock (Miami University) provided valuable advice during development and testing of these labs. Gretchen Webb-Kummer (Modesto Junior College) provided preliminary information on the Visual Sequence Editor program. Note W Supplementary materials for this article are available on JCE Online at http://jchemed.chem.wisc.edu/Journal/issues/1999/Oct/ abs1426.html.
Literature Cited 1. Reidhaar-Olson, J. F.; Sauer, R. T. Science 1988, 241, 53–57. 2. Mourad, G.; King, J. Plant Physiol. 1995, 107, 43–52. 3. Kochhar, S.; Finlayson, W. L.; Kirsch, J. F.; Christen, P. J. Biol. Chem. 1987, 262, 11446–11448. 4. Nowak, R. Science 1994, 265, 1352–1354. 5. Kramer, M. G.; Redenbaugh, K. Euphytica 1994, 79, 293–297. 6. Pennisi, E.; Williams, N. Science 1997, 275, 1415–1416. 7. For example, Zeidan, H. M.; Dashek, W. V. Experimental Approaches in Biochemistry and Molecular Biology; W. C. Brown: Dubuque, IA, 1996. 8. For example: Williamson, J. H. Am. Biol. Teacher 1997, 59, 164– 170. Beyer, A. Biochem. Educ. 1997, 25, 170–173. Hammond, J. B. W. Biochem. Educ. 1997, 25, 109–111. 9. Hamilton, R. G. J. Coll. Sci. Teach. 1997, 26, 351-354. 10. Beck, J. F.; Bunbury, D. L. DNA Sequencing; JCE Software 1997, 10 B No. 2. 11. Peters, K. Visual Sequence Editor, v. 1.1 (released Sep 1996). To download the program, go to the URL http://iubio.bio.indiana.edu. Under Software, select molecular biology, then mswin/, then mswinor-dos/, then vised11.readme. Follow the directions in the readme file to install the program, register, and obtain site licences. 12. Ito, Y.; Sasaki, T. Biosci. Biotechnol. Biochem. 1997, 61, 1270– 1276.
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