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.

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Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.

PCR (polymerase chain reaction), a commonly used molecular biology technique, has intrinsic limitations due to frequently encountered problems such as primer dimer formation and off-target amplification (1). To improve the specificity of PCR, TriLink's novel approach to "Hot Start" PCR employs chemically modified primers. CleanAmp™ primers are available as either CleanAmp™ Turbo or Precision and can be easily prepared by standard solid-phase oligonucleotide synthesis. These primers differ in the rate of release of the chemical protecting group thereby allowing for greater control of primer extension. We show that the presence of these primer modifications significantly reduces the amount of primer dimer and mis-priming products formed, relative to corresponding unmodified primers. Turbo primer modifications have shown great advantage in fast cycling and in multiplex reactions. In reverse transcription PCR and low copy number detection, Precision primers provide optimal performance. Overall, this unique approach to "Hot Start" activation offers valuable improvements to PCR performance. PCR conditions: 1X PCR buffer (10 mM Tris (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 ), Primers (0.5 µM), dNTPs (0.2mM), 5 copies HIV-1 gDNA, Taq DNA polymerase (1.25U), 50 µL. Thermal cycling conditions: 95 o C (10 min); [95 o C (40 sec), 56 o C (30 sec), 72 o C (1 min)]40X, 72 o C (7 min)

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BackgroundMultiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step.ResultsHerein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats.ConclusionsThe need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.