It is known that the Uracil-DNA Glycosylase can remove the basic residue of dU. Thus the DNA chain will further cleave at the point of dU when heating to 95℃
However, is there any method to cleave the dsDNA chain at point of dU in temperature
UDG cuts well at 37c on both ssdna and dsdna longer than 6 bases in length so I would expect it to cut slowly at lower temperatures. It is very difficult to heat inactivate and the 95c step will still leave some residual activity. the high temperature that you refer to is probably just to dissociate the dsdna prior to pcr as a common use of this enzyme is to remove possibilities of pcr contamination
Thanks! Paul Rutland yeah, to totally cleave the chain I 'm going to use certain con of NaOH or endonuclease to breake the AP site generated by the UDG
Solved. After the AP site created by the UDG, there are 3 ways to cleave the AP site: basic Denaturation buffer (0.015M NaOH+3%brij 35), certain endonuclease and denature temperature treatment
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology