Enzymatic synthesis of phosphorothioate oligonucleotides using restriction endonucleases
AssigneeGen-Probe Incorporated
InventorsDavid Bruce Lackey, Nanibhushan Dattagupta, Daniel Louis Kacian
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Abstract
The present invention relates to methods of synthesizing phosphorothioate oligonucleotides. In particular, it relates to the use of certain restriction endonucleases to cleave phosphorothioate oligonucleotides which contain restriction endonuclease recognition sequences. These restriction sequences facilitate the cleavage of relatively cleavage resistant phosphorothioate oligonucleotides thus facilitating their separation and purification after synthesis.
Claims20
A method of synthesizing at least one phosphorothioate oligonucleotide or phosphorothioate-containing oligonucleotide comprising the steps of:
- (a) providing a nucleic acid template, said template comprising a primer binding region and an oligonucleotide complementary region located 5' to said primer binding region,
- wherein said oligonucleotide complementary region comprises one or more subregions, each said subregion being complementary to an individual phosphorothioate or phosphorothioate-containing oligonucleotide to be synthesized,
- wherein said template is used to direct the synthesis of a primer extension product comprising a nucleic acid primer, an oligonucleotide product comprising said oligonucleotides, and one or more cleavage sites, said primer and said oligonucleotide product being separated by one of said cleavage sites and, when more than one oligonucleotide is to be synthesized, each pair of adjacent oligonucleotides is separated by one of said cleavage sites,
- wherein at least one of said cleavage sites is a restriction endonuclease recognition sequence selected from the group consisting of 5'-GGCC-3', 5'-GCGC-3', 5'-CCNGG-3', 5'-TCGA-3', and wherein N is any nucleotide and each nucleotide base of said recognition sequence is phosphorothioated;
- (b) contacting said template with said primer under conditions such that said primer hybridizes to the primer binding region of said template to form a template-primer hybrid;
- (c) incubating said template-primer hybrid in the presence of at least one DNA polymerase under conditions such that DNA synthesis occurs to form said primer extension product;
- and
- (d) cleaving said primer extension product such that said primer is separated from said oligonucleotide product and the oligonucleotides of said oligonucleotide product are separated from each other, wherein at least one cleavage is accomplished by contacting the primer extension product with an effective amount of a restriction endonuclease capable of recognizing said recognition sequence, said restriction endonuclease being selected from the group consisting of Hae III, HinP I, ScrF I and Taq.sup..alpha. I.
Dependencies are inferred from the claim text.