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Method for enzymatic synthesis of oligonucleotides

  • US 5,436,143 A
  • Filed: 12/23/1992
  • Issued: 07/25/1995
  • Est. Priority Date: 12/23/1992
  • Status: Expired due to Fees
First Claim
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1. A method for synthesizing an oligonucleotide of defined sequence, comprising the steps of:

  • (a) combining (1) an oligonucleotide primer and (2) a blocked nucleotide or a blocked nucleotide precursor that forms a blocked nucleotide in situ, in a reaction mixture in the presence of a chain extending enzyme effective to couple the blocked nucleotide to the 3'"'"'-end of the oligonucleotide primer such that a primer-blocked nucleotide product is formed, said blocked nucleotide comprising a nucleotide to be added to form part of the defined sequence and a blocking group attached to the nucleotide effective to prevent the addition of more than one blocked nucleotide to the primer;

    (b) inactivating the chain extending enzyme;

    (c) removing the blocking group from the 3'"'"'-end of the primer-blocked nucleotide product to form a primer-nucleotide product, whereby the reaction mixture contains unreacted starting materials, primer-nucleotide product and reaction by-products;

    (d) converting any unreacted blocked nucleotide in the reaction mixture to an unreactive form which is less active as a substrate for the chain extending enzyme than the blocked nucleotide;

    (e) repeating a cycle of steps (a) through (d) using the primer-nucleotide product of step (c) as the primer of step (a) for sufficient cycles to obtain a synthesized oligonucleotide of the defined sequence; and

    (f) cleaving the initial primer used in the first cycle from synthesized oligonucleotide.

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