DNA Synthesizer

kim at m44.unm.edu kim at m44.unm.edu
Thu Feb 14 14:49:29 EST 1991


This is my first try at posting in this group, so bear with me.  I believe that
this is a forum in which one may post even outlandish ideas and get
well-informed responses.  Well, here's one.                       
  I was thinking about DNA synthesis (as in making custom oligos) using terminal
transferase enzyme.  Although this enzyme is used for making homopolymer tails,
it should be possible to use it to make custom oligos on a solid matrix
(ideally a cheap dipstick) using an enzymatically-driven protect/deprotect
protocol.  For instance:
   Have a dipstick on which the first nucleotide of the reaction is attached by
biotin-avidin.  Dip into one reaction tube containing terminal transferase and
a solution of a single 3'-protected nucleotide triphosphate species in an 
appropriate buffer system.  Let incubate for a short time.  
   Wash the dipstick with saline, or some such, to get rid of residual enzyme
carry-over.
   Transfer to the de-protection reaction.  Incubate.
   Wash again.
   Transfer to the next nucleotide-enzyme reaction tube.
   Repeat until the oligo is complete.
   Release the finished (and 5'-biotinylated) oligo.

It is clear that the protect/deprotect system is the problem here.  What could
be used?  I was entertaining the idea of having 3'-phosphorylated nucleoside
tripohosphates as the protected species, and then using alkaline phosphatase to
deprotect.  Would it be useful to be able to make small amounts of custom oligos
on a benchtop using standard nucleic acid enzymology?

I would love to hear any ideas on this subject.   Daniel Kim



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