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 othe r than the restriction endonucleases in an in situ restriction enzyme diges t of chromosomes may induce G-like banding patterns, To determine whether f actors other than DNA base composition play a role in determining restricti on enzyme induced bands, we investigated the effect of reaction buffers alo ne or in the presence of heat inactivated enzymes. Our results show that en zymes such as AluI, Real, and MspI become inactivated during 3-24 hr incuba tions at 37 C and that reaction buffers alone failed to produce G-like band s when inactive endonucleases were included.