| 摘要 |
Provided herein are methods for ligase mediated nucleic acid replication and amplification of oligo- and probes containing substituted ligase components, particularly substituted ligase cofactors, substituted oligo- and probe acceptors, substituted oligo- and probe donors, substituted adenylated oligo- and polynucleotide donor intermediates carrying thermolabile group or groups. The substituted ligase components are not active until Hot Start activation step converts them into unsubstituted or natural ligase components, which fully support ligase reaction. The described methods are readily applied to ligation-based assays, especially utilizing Ligase Chain Reaction (LCR), for detection of a nucleic acid sequence where the use of the substituted ligase components improves an overall efficiency of LCR, increase discrimination between matched and mismatched templates and reduces or eliminates appearance of false positive signal. Furthermore, the use of the substituted ligase components reduces or eliminates the false positive signal originated from the template independent and blunt-ended ligation. |
