Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Abstract

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4′,6‐diamidino‐2‐phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species‐specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.

Lack of correlation between AT frequency and genome size in higher plants and the effect of nonrandomness of base sequences on dye binding

“…(1), but with AT ⅐ 2C instead of 2C. However, this seems not to be the case (6). Rather, a curvilinear relation exists in the form…”

“…sativum "Viktoria, Kifejtö Borsó" was used as the primary standard. The values for the genome size and AT frequency are based on reference (6). For the genome size, a value of 2C ϭ 9.07 pg is used.…”

“…(6) and (7) using a value of 59.5% for human leukocytes (6,13) and an assumed binding site length for DAPI of n ϭ 4. According to Table 1 in Doležel et al (6), the PI peak ratio of Homo sapiens leukocytes relative to the primary standard P. sativum is R_PI leuco ϭ 1/1.295 ϭ 0.772. Analogously, the DAPI peak ratio is R_DAPI leuco ϭ 1/1.432 ϭ 0.698.…”

“…According to eqs. (6) and (7), the DF for both dyes should be different. Moreover, it should be possible to calculate the unknown binding site length n DAPI from the known value n HO by the simplified relation *Binding site lengths n ϭ 4 (first line, regular characters) and n ϭ 5 (second line, italic characters) for different organisms calculated from the base sequence compared with a random base distribution of the same AT frequency.…”

“…Doležel et al (6) showed that base-specific fluorescence is not proportional to the base content, but rather follows a curvilinear relation. Provided that the bases in the genome are distributed randomly, a formula is given for the relation between base frequency and fluorescence intensity (8,9).…”