Banding patterns in human chromosomes: Production by proteolytic enzymes

VARIOUS techniques are used to produce banding patterns in human chromosomes, using trypsin68, pronase2, and a-chymotrypsin3. We have used trypsin and a-chymotrypsin as banding agents, and have found that pepsin also may be similiarly employed. These enzymes are highly efficient banding agents; however, the majority of metaphase preparations are readily ‘overtrypsinized’, so that the chromosomes appear fuzzy. This may be due to the action of the enzyme on the compacted chromosome, loosening the chro- monema and producing a lampbrush effect. In addition, when slides are dipped into a 0.25 percent trypsin solution, lymphocytes and fragments of debris are loosened and some are deposited over the metaphases. To overcome the drawbacks of trypsin processing we have modified Seabright’s method”. Before trypsin is applied, the slides are placed in 50 percent methanol for 20 minutes, washed in running tap-water for 10-15 minutes, then placed in 3:1 methanol:acetic acid for 2 hours and in absolute methanol for 2 hours. They are allowed to air dry. We use a 0.1 percent solution of trypsin (Grand Island Biological Co., New York, N.Y.; 0.25 percent solution, diluted with isotonic saline), or trypsin (2 x lyophilized; supplied by Worthington Biochemical Corp., Freehold, N.J.). The trypsin solution is applied for 10-30 seconds at room temperature, and then the slides are rinsed with isotonic saline. They can be stained with Leishman’s stain as described by Sea- bright or with Giemsa stain at a similar concentration. Slides previously stained with a quinacrine derivative, for fluorescence band studies, can be used for trypsin banding after removal of cover slips, washing, and refixing with 3:1 methanol:acetic acid as described.