Using the Comet Assay to Assess Genotoxic Damage
The single cell gel electrophoresis, or comet, assay is a powerful tool used by molecular biologists and toxicologists to visualize and quanitfy sublethal DNA damge in free cells or disaggregated tissue cells in any living organism. In essence, nucleated cells are collected from an organism (blood, hepatocytes, hemocytes, leucocytes, etc.) and embedded in a thin agar layer-coated microscope slide. The cells are then lysed, placed in an alkaline buffer where the DNA unwinds revealing any breaks in the single strands, and electrophoresed for a short time; this stretches out the DNA from the nucleoid. Staining and visualization reveals a comet-shaped cell. The length of the tail is directly proportional to the amount of damage, and can therefore be quantitatively or qualitatively evaluated. Singh and Tice, 1988; Olive et al, 1992.  This method is relatively simple, quick,inexpensive, and extremely sensitive making it ideal for use in the classroom. The activity will introduce cutting edge scientific technology to students.
Objectives
Students will:
Learn basic molecular biology laboratory principles and skills.
Quantify and analyze DNA damage to Chesapeake Bay organisms.
Begin to understand that toxic damage is not always evident Gain insight into DNA structure.
Materials
a source of nucleated cells: oyster hemolymph, fish or reptile blood ( F. heteroclitus, M. terrapin are good choices).
Trevigen CometAssay™ Silver Staining Kit, 200 Samples #4254-200-K
Plain microscope slides 18 x 50 mm
cover glasses
Low melting temp agar (Sigma #9414)
Normal melting temp agar
micropipettors and tips
Compound microscopes, 200x
30 % hydrogen peroxide working solutions: lysing, physiological buffer, neutralization buffer, electrophoresis buffer (see http://www.kineticimaging.com (Kinetic imaging) and http://cometassay.com/Files/raytice.doc (Ray Tice's protocol) for complete protocols and lab recipes
Gel electrophoresis system including power supply
Lab supplies: forceps, DI water, non latex gloves, etc.

Procedure
1.  Collect cells from organism.
2.  Incubate 10 uL cells in 0,10,25, and 50 micromolar hydrogen peroxide for 1 hour at room temp (22-25 C)in the dark 3.  Perform comet assay using 20 uL of each cell culture (see Tice protocol under materials).
4.  Score cells either qualitatively (see attached protocol), or quantitatively (for techno-savvy scientists) with available free image analysis software, http://rsb.info.nih.gov/ij/index.html and http://www.autocomet.com/software_download2.php
5.  Construct a dose response curve with results from the scoring assignment.
6.  Write up lab having student answer focus questions to guide their conclusions.  Note: as this is pretty involved science, lab groups (3-4) should work together to work through focus questions If doing this as a dry lab, use attached comet photo sheet to score in place of performing lab.
7.  Use a digital projector to go through scoring with the class in addition to using black and white handouts of photo data sheets.
References
Singh, N. P. and Tice, R. R. (1988). The single cell gel (SCG) assay. A New technique for quantitating DNA damage in individual cells. In Chauhan, P.S. and Chaphekar, S.B.(eds), Environmental Mutagenesis and Carcinogenesis, New Age International Ltd., Mumbai, pp.39-51.
Olive, P.L., Wlodek, D., Durand, R.E. and Banath, J.P (1992) Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp. Cell. Res., 198, 259-267.
State Standards
1.1.5- The student will explain factors that produce biased data (incomplete data, using data inappropriately, conflicts of interest, etc.).
1.2.3- The student will formulate a working hypothesis.
1.2.6- The student will identify appropriate methods for conducting an investigation (independent and dependent variables, proper controls, repeat trials, appropriate sample size, etc.).
1.3.3- The student will demonstrate safe handling of the chemicals and materials of science.(NTB)
1.3.4- The student will learn the use of new instruments and equipment by following instructions in a manual or from oral direction.(NTB)
1.4.1- The student will organize data appropriately using techniques such as tables, graphs, and webs (for graphs: axes labeled with appropriate quantities, appropriate units on axes, axes labeled with appropriate intervals, independent and dependent variables on correct axes, appropriate title).
1.4.2- The student will analyze data to make predictions, decisions, or draw conclusions.
1.4.9- The student will use analyzed data to confirm, modify, or reject an hypothesis.
1.5.1- The student will demonstrate the ability to summarize data (measurements/observations).
1.5.2- The student will explain scientific concepts and processes through drawing, writing, and/or oral communication.
1.6.2- The student will use computers and/or graphing calculators to perform calculations for tables, graphs, or spreadsheets.(NTB)
3.1.1- The student will be able to describe the unique characteristics of chemical compounds and macromolecules utilized by living systems.
3.1.2- The student will be able to discuss factors involved in the regulation of chemical activity as part of a homeostatic mechanism.
3.2.2- The student will conclude that cells exist within a narrow range of environmental conditions and changes to that environment, either naturally occurring or induced, may cause changes in the metabolic activity of the cell or organism.
3.3.4- The student will interpret how the effects of DNA alteration can be beneficial or harmful to the individual, society, and/or the environment.
3.5.3- The student will investigate how natural and man-made changes in environmental conditions will affect individual organisms and the dynamics of populations.
Lesson Resources
: Comet Lab
Comet PowerPoint