Chemically Modified Primers for Improved Hot Start PCR

DESCRIPTION (provided by applicant): The polymerase chain reaction (PCR) is a widely used technique for the amplification, manipulation and/or detection of DNA. Herein we propose the further development of a novel "Hot Start" PCR strategy which may have potential utility in the amplification of low-copy-number DNA. 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 further explore the feasibility of using synthetic oligonucleotide primers which contain modification(s) at the 3'- end. This modification, which can easily be introduced with a reagent that is compatible with conventional DNA synthesizers, is intended to prevent 3'-extension prior to a "Hot Start" preheating step. In that step, the modification is intended to undergo thermally-induced fragmentation to the corresponding unmodified oligonucleotide, which can then be recognized and extended by DNA polymerases. In Phase I, we demonstrated that the modified primers displayed the desired "Hot Start" fragmentation properties and that these primers could not be successfully extended by DNA polymerases prior to a preheating step. In Phase II, we plan to further develop the utility of these chemically-modified primers in PCR-based applications by optimizing the "Hot Start" properties. Chief among the envisioned advantages of the proposed new method for "Hot Start" PCR include its use with any thermostable DNA polymerase and buffer-pH. 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, as in all of these applications there are serious adverse consequences of false negatives and false positives. The polymerase chain reaction (PCR) is a widely used technique for the amplification, manipulation and/or detection of DNA. With the increasing shift towards the use of PCR for diagnosis, where accuracy of detection is of the utmost importance, several "Hot Start" PCR strategies have been commercialized to further improve the accuracy. Herein we propose the further development of a novel "Hot Start" PCR strategy which may have potential utility in the amplification of low-copy-number DNA.