Quantitative Microbiology 2, 69±79, 2000
# 2001 Kluwer Academic Publishers. Manufactured in The Netherlands.
Statistical Analysis of the Comet Assay Using a
Mixture of Gamma Distributions
BRYAN E. SHEPHERD, G. BRUCE SCHAALJE,
230 TMCB, Department of Statistics, Brigham Young University, Provo, UT 84602, USA
MICAH J. SMITH, BYRON K. MURRAY, KIM L. O'NEILL*
751 WIDB, Department of Microbiology, Brigham Young University, Provo, UT 84602, USA
*Corresponding author: tel.: +801 378 2449; fax.: +801 378 9197; e-mail: firstname.lastname@example.org
Received April 9, 2000; Accepted July 20, 2000
Abstract. Tail moments in the single cell gel electrophoresis (comet) assay usually do not follow a normal
distribution, making the statistical analysis complicated. Researchers have used a wide variety of statistical
techniques in an attempt to overcome this problem. In many cases, the tail moments follow a bimodal distribution
that can be modeled with a mixture of gamma distributions. This bimodality may be due to cells being in two
different stages of the cell cycle at the time of treatment. Maximum likelihood, modi®ed to accommodate
censored data, can be used to estimate the ®ve parameters of the gamma mixture distribution for each slide. A
weighted analysis of variance on the parameter estimates for the gamma mixtures can be performed to determine
differences in DNA damage between treatments. These methods were applied to an experiment on the effect of
thymidine kinase in DNA damage and repair. Analysis based on the mixture of gamma distributions was found to
be more statistically valid, more powerful, and more informative than analysis based on log-transformed tail
Key words: tail moment, comet assay, single cell gel electrophoresis assay, bimodal distribution, maximum
The single cell gel electrophoresis assay (comet assay) is an increasingly popular method
for detecting and comparing nuclear DNA damage and repair. There are many advantages
and utilities to this technique (Fairbairn et al., 1995).
The ``tail moment'' is the most widely used measurement of DNA damage in the comet
assay. It is calculated for individual cells as the product of the mean displacement and the
percentage of the total DNA in the tail (Hellman et al., 1995). In almost all cases the tail
moments of a sample of cells do not follow the normal distribution, and often the variance
of tail moments between samples of cells exposed to different treatments is unequal. This
causes dif®culty when performing statistical analyses because these assumptions are
necessary in order for t-tests or analyses of variance (ANOVA) to be valid.
One solution has been to perform a logarithmic transformation on the tail moments,
making their distributions more normally shaped and their variances more homogeneous.