Chemically Modified dNTPs as a General Approach to Improved Hot Start PCR

描述(申请人提供)：聚合酶链式反应(PCR)是一种用于扩增感兴趣的DNA序列的强大技术。该技术的不断进步包括实时聚合酶链式反应技术和逆转录聚合酶链式反应技术的发展。随着这些和其他允许对感兴趣序列进行准确定量的技术的出现，对该技术的精确度的改进存在持续的需求。在此，我们建议进一步发展一种新的热启动PCR策略，该策略可以通过减少不需要的扩增产物的数量来提高PCR的特异性。虽然已经开发了许多热启动聚合酶链式反应技术，但这些技术都没有利用化学修饰的合成脱氧核苷5‘-三磷酸(DNTPs)。本提案旨在进一步开发改良的dNTPs作为聚合酶链式反应中热启动激活的通用解决方案。预期这种热启动聚合酶链式反应的方法应该适用于现有的聚合酶链式反应技术，允许通过简单地用未修改的dNTP混合物替换相应的修改的dNTP混合物来与现有的聚合酶链式反应系统一起使用。此外，我们还建议进一步探索该技术在单核苷酸多态(SNP)检测和微阵列分析等应用中的应用。总体而言，我们建议开发一种新的热启动PCR方法，该方法将为核酸扩增提供更高水平的特异性，并具有在许多基于PCR的平台上使用的灵活性。

DESCRIPTION (provided by applicant): 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.