We have been investigating strategies for novel methods of hot start for PCR. One such strategy involves the sequestering of a critical component of the reaction until the reaction temperature is sufficient to ensure selective annealing of PCR primers. We have investigated reversible precipitates of Mg as a method of sequestering Mg2+ ions, which are a critical component of PCR. Many PCR analyses benefit from or require a hot start, particularly those with rare targets or those with compromised DNA templates, such as diagnostic or forensic analyses. In a normal (room temperature) start, the primers can bind to non-specific sequences, or each other, during reaction setup, which can result in the amplification of competitive or inhibitory products. Current methods of achieving a hot start are tedious, expensive, and/or unreliable. We have identified a precipitated form of Mg that is solublized by thermocycling, but is stable enough at room temperature to inhibit polymerase activity. We have also determined that PCR reactions performed with this reversibly precipitated Mg have the benefit of a hot start. We are proposing to simplify and optimize this reversible precipitate technology for standard PCR and extend it to specialized PCR applications, multiplex PCR, Long and Accurate PCR, and even those applications that use other Mg2+ ion dependent enzymes, such as cDNA synthesis by reverse transcriptases.
