What is the role of the Klenow DNA Pol in End Repair?

[odile]

Hi everyone,

Please forgive my naive questions, I am just begining to work with NGS. I need to modify the blunt end step to incorporate repair enzymes (UDG and Endo VIII as described in one of the Neanderthal NGS papers).
I try to understand exactly what is going on during the blunt end and I wonder why we need the Klenow pol since the T4 DNA Pol has a 3'-5' exonuclease activity? Is it to fill up nicks?

[bbeitzel]

The 454 Standard Library prep only uses T4 DNA pol and PNK during the end polishing step. The Klenow comes in during the fill-in step to add the adapter sequence to the other end of the library fragments (since the adapters are not phosphorylated.) The strand displacement activity of Klenow is necessary for the fill in reaction (nick translation.) I don't know off hand, but I am guessing that T4 DNA pol does not have strand displacement activity.

The 454 Rapid library prep uses different biochemistry with Taq and phosphorylated primers, and does not use Klenow.

Hope this helps some.

***Edit***
Sorry for this reply. I saw your post in the New Posts section and didn't notice that your initial question was for Illumina library prep.

[ECO]

Moving to sample prep.

[odile]

Sorry, I should have mentioned it was for the Illumina library. That's the reason why I had put it in the Illumina forum. There is no need of Bst with Illumina. I actually spoke to NEB and they released a new kit for Illumina library prep that doesn't incorporate Pol I (Klenow) at all. Nobody seems to know why the blunt end protocol from Illumina uses 2 enzymes that do exactly the same thing

[HESmith]

Klenow is much less expensive per unit than T4 polymerase, but is less efficient at 3'->5' exo activity. Mixing the two provides the most economical method for obtaining the desired activity - although, given the price of the kits, that seems the least of their concerns:-).

[odile]

That makes sense. The tech support from Illumina responded that T4 pol was better for removing the 3' overhangs but Klenow is better for filling in 5' overhangs. Not sure this is accurate. When I look at enzyme info from NEB, it says that both enzymes incorporate 10nM of dNTP in 30 min at 37 degrees.

[odile]

I have another question
What happens when the T4 DNA Pol faces an abasic site?
Does it stop extending?

Oh and one more: if you believe that your overhangs (on both sides) contain a lot of damaged bases, wouldn't it be preferable to use Pol I alone to create blunt ends on both sides (no elongation)?