Chapter 22 Looking at Diploid Interphase Chromosomes

Using this two-color in situ hybridization protocol, we can resolve two signals using a minimum probe size of 7.8 kb, which are separated from each other by 25 kb. We would be unable to resolve these signals if they were the same color. Shorter probes have been used in mammalian cells (Xing et al., 1993). Presumably, this problem will be solved with subtle improvements in technique, and perhaps, by sacrificing preservation to some extent. The question now is, what is the protocol good for? The results of the hybridization are clear enough and what we see is at least consistent with what we expect. The signal is in a position in the nucleus indicative of its position on the chromosome and there are one or two signals in each nucleus. When two probes that lie near each other on the molecular map are chosen, they appear close together in the nucleus. However, interpretation of all DNA in situ hybridization techniques poses several problems. The first is that conditions used to denature nucleic acids might alter cellular structures, so material must be fixed. Fixation artifacts may be undetected until a large number of data are produced and may be subtly different in different stages of the cell cycle as the level of DNA modification and the complement of associated proteins changes. It would be ideal to find a procedure that allows visualization to DNA that does not require denaturation or fixation. Simultaneous visualization of RNA and DNA has been achieved by others (Xing et al., 1993), but we have yet to attempt it.