Exonuclease digestion of human chromosomes for in situ hybridization and R-banding.

In situ hybridization of human chromosomes appears as one of the most attractive methods of gene mapping since it enables the locus being looked for to be directly visualized. This technique however includes a critical step which is the denaturation of chromosomal DNA by different procedures such as heat, alkali or acid treatment (1). These affect the chromosome structure and decrease the good guality of the posthybndization chromosome banding which is essential to identify chromosomes and locate the probe. Recently, Schmidt (2) reported that use of exonuclease HI was a more gentle method to digest DNA on polytene chromosomes and to obtain a good chromosomal hybridization. This procedure has been applied on human chromosomes. The results are shown in figure 1. They showed the hybridization of the 3/3hydroxysteroid dehydrogenase (HSD /33) on human chromosomes. The cDNA coding for HSD /33 hybndes on the pl3 band of chromosome 1. This localization is the same as previously reported (3) with chromosomes denatured by alkali treatment. The data showed that exonuclease m is effective in digesting one of the two strands of human chromosomal DNA rendering the other strand available for in situ hybridization. Moreover the chromosomal structure is much better preserved and R-banding that is used after the hybridization procedure is of high quality and obtained with more constancy. Figure 2 shows for example one chromosome 2 with R-banding after alkali treatment compared to exonuclease treatment.