Reply

Abstract

We thank Drs. Doležel, Voglmayr, Bartoš, and Greilhuber for their help in correcting a mistake in our calculation of the DNA content of the TRBC standard used in the studies published in Cytometry (1991;43:2–11). My main field of study is engineering, so I feel somewhat like a fish (trout) out of water when addressing some of the points raised in their letter. A failure to specify the species of trout used in the study. Improper calculation of the average molecular weight of the nucleotides. A wrong assessment of the human genome size. The difference in the ratio of human versus trout between our observations and the values published by others. The species of trout used in this study was clearly specified on page 4 in Materials and Methods as Salmo gairdnerii irideus obtained from the U.S. Fish Hatchery, Erwin, Tennessee. The molecular weights of the nucleotides were improperly calculated and are approximately 10% too high. They were based on the incorrect formulas for the nucleotides including the backbone (Table 2). The literature nomenclature is somewhat confusing to nongeneticists when discussing the size of the human genome. I was uncertain whether Mb referred to mega bases or mega base pairs. I relied on the information published about the Human Genome Project by the U.S. Department of Energy. On their Web site (http://www.ornl.gov/hgmis/), they claim the following as one of their goals for the Human Genome Project: “determine the sequences of the 3 billion chemical base pairs that make up human DNA.…” It has been my understanding as a layman in the field that this number is used quite often as an approximation to the genome size in base pairs. Therefore I assumed that the published values in Mb meant mega bases, not mega base pairs. The values used in our paper of approximately 6 billion nucleotides corresponds to 3 billion base pairs. The differences between our values of the ratio of human to trout can be explained by a number of factors such as species differences and staining. We found this ratio to be constant in more than 4,000 human specimens run in four different laboratories. We used 4,6-diamidino-2-phenylindole as the DNA stain at a concentration of 10 μg/ml. We believe that the values referred to in the letter by Doležel and colleagues were derived with propidium iodide as the stain. We would be happy to supply some of our TRBC to anyone interested in resolving this issue. In summary, we believe that the values published in our paper are in error by approximately 10% due to the wrong formulas used for the nucleotides. As we mentioned in the paper, the “effective chromosome size” used for these calculations excludes repetitive DNA such as that found on the short arms of the acrocentric chromosomes. This error may be many times the value of the calculation error and still needs to be determined as the Human Genome Project proceeds. Richard A. Thomas*, * RATCOM, Inc. Miami, Florida.