5amp;prime;-thio phosphate directed ligation of oligonucleotides and use in detection of single nucleotide polymorphisms

摘要

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.