Chemically modified primers for improved hot start PCR

Abstract

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)

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

“…However, while this approach has been shown to be a completely independent alternative Hot Start method, it potentially may provide synergistic advantages when used in conjunction with alternative Hot Start technologies. Overall, the described modified primer technology ( 95 ) represents an orthogonal approach to Hot Start activation in PCR, which may provide alterative benefits to PCR that have not been possible with current Hot Start DNA polymerase-based approaches.…”