SBIR-STTR Award

The polymerase chain reaction (PCR) is a powerful technique used to amplify a DNA sequence of interest. Continual advancements to the technique have included development of reverse-transcriptase PCR and real- time PCR technologies. With the advent of these and other technologies that allow for accurate quantitation of a sequence of interest, there are continual needs for improvements to the accuracy of the technique. Herein we propose the development of a novel Hot Start PCR strategy which may improve the specificity in PCR by reducing the number of undesired amplification products. Although numerous Hot Start PCR technologies have been developed, none of these utilize chemically-modified synthetic deoxynucleoside triphosphates (dNTPs). The present proposal aims to further explore the feasibility of using modified dNTPs that inhibit primer extension until a "Hot Start" preheating step is performed. It is anticipated that this approach to Hot Start PCR should be amenable to existing PCR technologies. This may allow application for use with existing PCR systems, by simple substitution of the unmodified dNTP mix with the corresponding modified dNTP mix. Overall, we propose the development of a novel approach to Hot Start PCR that will offer an added level of specificity to nucleic acid amplification. The polymerase chain reaction (PCR) is a molecular biology technique that is becoming routinely used in the field of molecular diagnostics. In this field, accurate identification of DNA sequences is imperative for reliable diagnosis of disease. To further improve the accuracy of the PCR technique, we propose the investigation of a novel Hot Start PCR approach, in which the dNTP substrates are chemically modified.

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The polymerase chain reaction (PCR) is a powerful technique used to amplify a DNA sequence of interest. Continual advancements to the technique have included development of real-time PCR technologies and reverse-transcription PCR. With the advent of these and other technologies that allow for accurate quantitation of a sequence of interest, there are continual needs for improvements to the accuracy of the technique. Herein we propose the further development of a novel Hot Start PCR strategy which may improve the specificity in PCR by reducing the number of undesired amplification products. Although numerous Hot Start PCR technologies have been developed, none of these utilize chemically-modified synthetic deoxynucleoside 5'- triphosphates (dNTPs). The present proposal aims to further develop modified dNTPs as a general solution for Hot Start activation in PCR. It is anticipated that this approach to Hot Start PCR should be amenable to existing PCR technologies, allowing for use with existing PCR systems, by simple substitution of the unmodified dNTP mix for the corresponding modified dNTP mix. In addition, we propose to further explore the utility of this technology in applications, such as single nucleotide polymorphism (SNP) detection and microarray analysis. Overall, we propose the development of a novel approach to Hot Start PCR that will offer an added level of specificity to nucleic acid amplification, with the flexibility for use in a number of PCR-based platforms.

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