The effects of enzyme inactivation and incubation buffer on digestion in situ with restriction endonucleases

Previous studies have shown that components of the incubation reaction other than the restriction endonucleases in an in situ restriction enzyme digest of chromosomes may induce G-like banding patterns. To determine whether factors other than DNA base composition play a role in determining restriction enzyme induced bands, we investigated the effect of reaction buffers alone or in the presence of heat inactivated enzymes. Our results show that enzymes such as AluI, RsaI and MspI become inactivated during 3-24 hr incubations at 37 C and that reaction buffers alone failed to produce G-like bands when inactive endonucleases were included.