I'm trying to do Klenow extension of DNA that is immobilized to a bead using a primer that should anneal to the DNA on the bead and have overhang and then use Klenow to extend. I'm not having much success and was wondering if anyone had experience with a similar experiment. I'd prefer not to use a biotinylated strategy.
Bryan, What you are describing is essentially what goes on in an Illumina sequencing reaction. The only real difference is the polymerase, but Klenow is a very well behaved enzyme and should work. I am betting that the most likely problem is with the primer annealing rather than the enzyme extension. Is there anything about the immobilization that might block access to the oligonucleotide? I would raise the temperature (i.e. 60oC) and then slowly lower it to the hybridization temperature to favor access. Are you sure that the DNA is really immobilized on the beads? Is there a high enough concentration of the primer to drive the hybridization kinetics? Hybridization to immobilized templates is slower than in solution.
As a secondary test, I would consider doing the annealing in solution first to prove that all other components of the reaction are working well.
Thanks! We're using the IDT annealing conditions and everything appears to work in solution. We're raising the temp to 95 and then doing a slow cool. We've tried using a fluorescent oligo and annealing that to the bead and then looking for binding that way. I'm not sure what the critical concentration of the primer should be to drive hybridization kinetics, but I feel the fluorescent oligo shows that?
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