Chromosome Preparation and High Resolution Banding Techniques. A Review

Abstract

High resolution banding techniques enable detection of chromosome rearrangements even within major bands. Banded chromosomes prepared for light microscopic studies of intact metaphase plates are, however, highly modified structures compared with native chromosomes, and the high resolution banding techniques only seem possible because the following methods were standardized and combined. The use of colcemid, which prevents formation of the spindle and thereby collects cells at the metaphase-anaphase border, is routinely used for chromosome preparations. For high resolution banding studies, short exposure time and concentrations near the threshold value have been recommended by several authors. Several agents interfere with chromosome contraction processes, but only a few have had a lasting influence on high resolution banding studies. The most used agents are ethidium bromide, actinomycin D, and Hoechst 33258, which all partially inhibit chromosome contraction. Treatment with hypotonic solutions induces swelling of animal cells, and the methanol in the fixative denatures and precipitates protein by dehydration. The acetic acid coagulates nucleoproteins and causes swelling of the cells. The fixative penetrates the cells rapidly and preserves the chromosome structure. To obtain long segmented chromosomes suitable for high resolution banding hypotonic treatment with .075 M KCl, frequent changes of fixative and overnight fixation at 4°C have been recommended. The use of cell synchronization, 5-bromodeoxyuridine incorporation into DNA, and fluorochrome-photolysis Giemsa (FPG)-staining have improved the quality of high resolution banding. Synchronization techniques, which select for lymphocyte populations in early divisions, provide excellent materials for chromosome preparations and induction of high resolution banding. The banding techniques seem to enhance differences already present in the chromosomes, and the differential Giemsa staining has recently been explained by interactions between the hydrophobic dye complex, the supercoiled DNA helix, and the denaturated histone core of the nucleosomes.