Chemically Modified Primers for Improved Hot Start PCR

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to provide a new and significantly improved "hot start" method for carrying out the polymerase chain reaction (PCR) for amplification, manipulation, and/or detection of DMA. "Hot start" PCR is widely used in molecular biology and biotechnology, and is also being applied to various types of genetic testing, clinical diagnostics, blood-screening, forensics and biodefense. These diverse health-related applications are predicated on the high specificity by which "hot start" PCR can amplify low-copy-number target DMA. Several strategies for conducting "hot start" PCR have been commercialized; however, none of these utilize chemically-modified synthetic oligonucleotides that offer significant potential advantages relative to currently available methods. The present proposal aims to assess the feasibility of using synthetic oligonucleotide primers which have a single phosphotriester-modification at the 3' end. This modification, which can be easily introduced with a reagent that is compatible with conventional DNA synthesizers, is intended to prevent 3' extension of the primer prior to a "hot start" step. In that step, the phosphotriester modification is intended to undergo thermally-induced, non-hydrolytic, fragmentation to generate a natural, unmodified internucleotide linkage that can then be recognized and extended by DNA polymerases. Chief among the envisaged advantages of the proposed new method for "hot start' PCR are its use with any thermal-stable DNA polymerase and buffer-pH, as well as greater reproducibility due to absolutely homogenous reaction conditions. It is also proposed that this new method when used in combination with currently available "hot start" DNA polymerases may provide greater levels of specificity and reliability for genetic testing, clinical diagnostics, blood-screening, forensics and biodefense since in all of these applications there are especially serious, adverse consequences of "false negatives" and "false positives". Synthetic routes are presented, as is the use of a simplified, 3'-modified-primer/duplex-DNA-target model system for studies using analytical separations by HPLC and PAGE, Each of the specific aims has a corresponding contingency plan to hopefully ensure completion of feasibility in a 6-month timeframe.