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

化学修饰 dNTP 作为改进热启动 PCR 的通用方法

• 批准号: 7634464
• 负责人: Natasha Paul
• 金额: $ 36.47万
• 依托单位: TRILINK BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7634464
• 关键词: 2' -Deoxythymidine; 2' -deoxyadenosine; Biochemical Reaction; Biomedical Research; DNA; DNA Microarray Chip; DNA Sequence; DNA-Directed DNA Polymerase; Deoxycytidine; Deoxyguanosine; Development; Diagnostic; Evaluation; Evolution; Fluorescein; Fluoresceins; Goals; Government; Grant; Hand; In Vitro; Investigation; Knowledge; Label; Lead; Ligation; Measures; Microarray Analysis; Modification; Molecular; Nucleic Acids; Nucleosides; Oligonucleotide Primers; Performance; Persons; Phase; Phase I Clinical Trials; Phase II Clinical Trials; Polymerase; Polymerase Chain Reaction; Polymorphism Analysis; Preparation; Prevention; Primer Extension; Process; Property; Protocols documentation; Reaction; Reverse Transcription; Route; Series; Shipping; Ships; Single Nucleotide Polymorphism; Small Business Technology Transfer Research; Solutions; Specificity; Structure; Surface; System; Techniques; Technology; Temperature; Therapeutic; Time; Tube; Universities; Work; analog; base; cold temperature; cost; deoxyguanosine triphosphate; dimer; flexibility; improved; interest; novel; novel strategies; nuclease; nucleobase; nucleobase analog; research study; seal; thermostability; tripolyphosphate

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.

描述（由申请人提供）：聚合酶链反应（PCR）是一种用于扩增目标DNA序列的强大技术。该技术的不断进步包括实时PCR技术和逆转录PCR的发展。随着允许准确定量感兴趣序列的这些和其它技术的出现，持续需要改进该技术的准确性。在此，我们提出了一种新的热启动PCR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。尽管已经开发了许多热启动PCR技术，但是这些技术都没有利用化学修饰的合成脱氧核苷5 '-三磷酸（dNTP）。本提案旨在进一步开发修饰的dNTP作为PCR中热启动激活的一般解决方案。预计这种热启动PCR的方法应适用于现有的PCR技术，允许与现有的PCR系统一起使用，通过简单地用未修饰的dNTP混合物取代相应的修饰的dNTP混合物。此外，我们建议进一步探索该技术在应用中的效用，如单核苷酸多态性（SNP）检测和微阵列分析。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的平台中使用的灵活性。