RNase H Hot-Start Assays for Specific Oligonucleotide Amplification

The primer is pre-modified with a blocking group at the 3'-end before the PCR reaction begins. This blocking group prevents premature primer extension at lower temperatures while allowing the primer to hybridize to the target sequence. The blocking group is subsequently removed by a specific enzyme (such as a phosphatase or glycosylase) only after proper hybridization has occurred, thereby enabling extension only under appropriate conditions and preventing non-specific amplification and primer dimer formation.

The invention extracts the hot-start control mechanism from the DNA polymerase enzyme itself and transfers it to the primer molecule through the blocking group modification. This eliminates the need for reversibly inactivated polymerases, antibody-polymerase complexes, or other complex hot-start mechanisms. The simplified system uses only the modified primer and a specific removing enzyme, reducing reaction mixture complexity while maintaining amplification specificity.

The blocking group is placed at a specific local position (the 3'-end) of the oligonucleotide primer, rather than modifying the entire molecule. This localized modification allows the rest of the primer to maintain its standard sequence and structure, facilitating synthesis. The blocking group can be incorporated during standard oligonucleotide synthesis using modified phosphoramidites, and its removal is achieved through a specific enzymatic reaction that acts only on the blocking group, not the entire oligonucleotide, thereby balancing specificity enhancement with manufacturing feasibility.