klenow/t4 and dNTPs - question (Nov/13/2008 )
I am trying to understand a blunt end reaction that takes place with klenow and t4 dna polymerases and dNTPs. These three are mixed together in one tube along with dna.
I have sheared genomic dna that needs to get end repaired using klenow and t4. As I understand so far, both polymerases have 3'->5' exonuclease activity so they can both be used to remove 3' overhangs. I heard Klenow has higher fidelity and is better at 5' fill-ins using its polymerase activity. The dNTPs will help prevent excessive exonuclease activity and they will create blunt ends.
Is there anything else that happens when these two polymerases are mixed together? Please let me know if this is right or if there is more to it than this - am I missing anything in understanding how this reaction takes place to create blunt ends on sheared dna?
Actually, which one of the two enzymes you need to use depends on the restriction enzyme you've used to digest your DNA. I personally never use both enzymes together and don't know of anyone that does. Klenow is said to work best with 5' overhangs, whereas T4 DNA Polymerase works efficiently with both 5' and 3' overhangs. High concentration (i. e. 100 μM) of dNTPs will prevent degradation of dsDNA due to the very high exonuclease activity of T4 DNA Polymerase.
Hope this helps!