| 摘要 |
Enzymatic synthesis of oligonucleotides is performed by the steps of: (a) combining a primer and a blocked nucleotide in the presence of a chain extending enzyme to form a primer-blocked nucleotide product containing the blocked nucleotide coupled to the primer at its 3'-end; (b) removing the blocking group from the 3' end of the primer-blocked nucleotide product; and (c) repeating the cycle of steps (a) and (b), using the primer-nucleotide product of step (b) as the primer for step (a) in the next cycle, for sufficient cycles to form the oligonucleotide product. Cycles may optionally include the step of converting any unreacted blocked nucleotide to an unreactive form which is substantially less active as a substrate for the chain extending enzyme. Cycles may also include the step of removing the blocking group from unreacted blocked nucleotide. This step is unnecessary, however, when the same nucleotide is added in two or more successive cycles. The synthetic cycles are preferably performed in a single vessel without intermediate purification of oligonucleotide product.
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