Many PCR analyses, particularly those with rare targets or those with compromised DNA templates (such as forensic analyses), benefit from a hot start. In a normal (room temperature) start, the primers can prime at non-specific sequences, or on each other, during reaction setup. The result can be competitive or inhibitory product produced at the expense of the desired product. In a hot start, a critical component is kept inactive or separate from the reaction until the temperature is at least 65C, at which temperature the primers are properly selective. Current methods of achieving a hot startare tedious, expensive, and/or have shortcomings. We propose to develop heat-labile chelators of Mg++ ion as hot-start reagents for PCR. The DNA polymerases used in PCR reactions are Mg++ dependant, and the Mg++ chelator EDTA inhibits PCR. The ideal hot-start chelator would be stable at room temperature in PCR reaction buffer for at least an hour, but will inactivate and release the Mg++ ion during the first PCR heat cycle, yet not inhibit the PCR reaction itself. To perform hot-start with these reagents, sequestered Mg++ ions (in the form of soluble Mg-chelate) would be added to PCR reactions in place of MgCI2 as a source of Mg++ ions. We propose three lead groups of compounds that have the structural characteristics of Mg++ chelators, and can be inactivated under controlled conditions.
Methods for hot-start PCR have proven to be impartant-- even critical-- for many PCR procedures, such as forensic analysis, food and blood product contamination assays. Although most PCR applications benefit from hot-start methods, they are underused because they add significant cost and effort, and they have inadequacies. The labile Mg-chelating reagents we propose to develop and test will be inexpensive, easy to use, and will likely be more effective mediators of hot-start than current methods. They may in fact be inexpensive enough to become standard reagents for PCR used by thousands of laboratories world wide. Such labile chelators may also have applications for other biotechnology and chemical processes.
