kim@m44.unm.edu (02/14/91)
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