Nucleic Acids Research Advance Access published online on September 16, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn575
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Methods Online |
Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance
Department of Research and Development, TriLink BioTechnologies, Inc., 9955 Mesa Rim Road, San Diego, CA 92121, USA
*To whom correspondence should be addressed. Tel: +1 858 546 0004; Fax: +1 858 546 0020; Email: npaul{at}trilinkbiotech.com
Received May 15, 2008. Revised August 22, 2008. Accepted August 26, 2008.
The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation 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 linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, 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 specificity and efficiency of nucleic acid target amplification.
Present address: Gerald Zon, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA