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AssigneeMolecular Staging, Inc.
InventorsRajanikanth Bandaru, Gyanendra Kumar
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The present invention provides a novel method for ligation of oligonucleotides containing 5′-phosphorothioates on complementary templates by the action of DNA ligases. This reaction is readily applied to the synthesis of a single stranded circular DNA containing a phosphorothioate linkage at the site of ligation junction. The efficiency of 5′-phosphorothioate directed ligation reaction by ATP dependent DNA ligase reaction is similar to conventional 5′-phosphate ligation. The utility of enzymatic ligation in probing specific sequences of DNA is also described. The present invention also provides a novel non-enzymatic ligation of 5′-phosphorothioates that has been applied to the synthesis of single strand phosphorothioate and phosphate circular DNA. A process for detecting the presence of a mismatch in an otherwise complementary pair of oligonucleotides is disclosed using an enzyme-based technique which shows the presence of a mismatch by failing to form a ligated single stranded DNA circle that can optionally be amplified using standard methods of rolling circle amplification.
A process for forming a single stranded circular oligonucleotide comprising contacting 3′-hydroxyl and 5′-phosphorothioate groups of an open circle oligonucleotide (OCO), having such groups at its 3′- and 5′-ends, with a bridging oligonucleotide (BO), said BO comprising sequences complementary to sequences at the 3′- and 5′-ends of said OCO, in the presence of a ligation catalyst that directs formation of a 3′-5′-phosphodiester linkage with the sulfur atom of said 5′-phosphorothioate group not present as a bridging atom thereby forming a single-stranded circular oligonucleotide.
Dependencies are inferred from the claim text.