title: Hot start PCR with heat-activatable primers: a novel approach for improved PCR performance.
abstract: 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(R) 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.
authors.affiliation: Department of Research and Development, TriLink BioTechnologies, Inc., 9955 Mesa Rim Road, San Diego, CA 92121, USA.
authors.forename: Alexandre V
authors.initials: AV
authors.lastname: Lebedev
authors.forename: Natasha
authors.initials: N
authors.lastname: Paul
authors.forename: Joyclyn
authors.initials: J
authors.lastname: Yee
authors.forename: Victor A
authors.initials: VA
authors.lastname: Timoshchuk
authors.forename: Jonathan
authors.initials: J
authors.lastname: Shum
authors.forename: Kei
authors.initials: K
authors.lastname: Miyagi
authors.forename: Jack
authors.initials: J
authors.lastname: Kellum
authors.forename: Richard I
authors.initials: RI
authors.lastname: Hogrefe
authors.forename: Gerald
authors.initials: G
authors.lastname: Zon
mesh_terms: D052160:Benzothiazoles; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry*; D004279:DNA, Viral / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003959:Diamines; D019281:Dimerization; D005456:Fluorescent Dyes; D015497:HIV-1 / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D009930:Organic Chemicals; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D009943:Organophosphorus Compounds; D011804:Quinolines; C434331:phosphoramidite; C098022:SYBR Green I; D004259:DNA-Directed DNA Polymerase
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