ddNTP to a 3'-end of DNA primer

Dmitry Mozzherin dim at pharm.som.sunysb.edu
Mon Jul 25 16:16:09 EST 1994


Hi,
I have a problem with inserting of dideoxynucleotides into 3'-end of DNA
primer. I should to receive only ddNMP-3'-ends without any 3'-OH ends. 
But if I use Klenov fragment (with 3'-5' exonuclease inhibited by AMP), or 
terminal transferase, I never have 100% yield. I could not use gel 
purifying, because I work with hooked template-primers with various length.
I had tried to digest free hydroxyl ends with 3'-5' exonucleases of Klenov
fragment, or T4 DNA polymerse, or exonuclease III. Klenov fragment and T4
DNA polymerase binds to deoxy-3'-ends (I think so) and could not work with
3'-OH ends. Exonuclease III works good, but it digest very well OH- and 
deoxy- ends. And I have nothing after it.
I would be very obliged, if somebody could help me with it.

With best wishes,
Dmitry Mozzherin  



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