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The polymerase chain reaction (PCR) is widely used for applications which require a high level of speci- ficity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biode- fense. Great improvements to PCR performance have been achieved by the use of Hot Start activa- tion strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3'-terminal and 3'-penultimate internucleotide link- ages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower tempera- tures that exist prior to PCR amplification. Further- more, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one- step reverse transcription (RT)-PCR and in real-time PCR with SYBR Green I dye and Taqman probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the speci- ficity and efficiency of nucleic acid target amplification.

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance