In Situ Hybridization of Meiotic Prophase Chromosomes
In preparations of highly condensed metaphase chromosomes, it is often difficult to resolve in situ hybridization signals of probes to nearby regions. This is in part, a result of traditional fixation procedures that use alcohol, acetic acid, and/or relatively strong ionic conditions, all of which can cause chromosome condensation. The limit of resolution may be improved by using free chromatin or DNA-halo preparations as described in Chapters 10 and 11 of this volume. An alternative method of detecting proximal DNA sequences is to use whole-mount, surface spread meiotic prophase chromosomes (1, 2), which have less condensed chromatin and a well defined axial core that is much longer than that of a metaphase chromosome. We have tested this system using two probes (3) against low-copy repeats, 5.5 map units apart on the X chromosome (4) as shown in Fig. 1. On metaphase chromosomes, these signals wereclose together and often overlapped. However, a distance of several micrometers separated the signals on pachytene chromosomes. This type of mapping is useful provided one keeps in mind that the chromatin is arranged in loops around a protein core, the synaptonemal complex (SC) (Fig. 2) (5), and that these loops are of considerable size relative to those of the metaphase chromosome. Thus, a sequence at the top of a 5-μm loop could appear some distance from the SC loop attachment point, depending on how the loop settles during chromosome preparation. Therefore, the size of the loop limits the resolution of the probes a few μm apart. However, if the probes are very close together (a few megabases), the decondensed chromatin permits resolving signals from probes to adjacent loci or to cosmid probes of different regions within the same gene.
KeywordsRecombination Rubber Boiling Styrofoam HEPES
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