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

基本信息

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
项目类别：
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&apos -Deoxythymidine 2&apos -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

项目摘要

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）是一种强大的技术，用于扩增感兴趣的DNA序列。该技术的持续进步包括开发实时PCR技术和反向转录PCR。随着这些技术和其他技术的出现，可以准确地定量感兴趣的顺序，因此有不断提高技术准确性的需求。在此，我们提出了一种新型热门PCR策略的进一步发展，该策略可以通过减少不希望的放大产品的数量来提高PCR的特异性。尽管已经开发了许多热启动PCR技术，但这些都没有使用化学改性的合成脱氧核苷5'-三磷酸盐（DNTPS）。本提案旨在进一步开发经过修改的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的方法应适合现有的PCR技术，从而可以与现有的PCR系统一起使用，并通过简单替换未修改的DNTP混合物来代替相应的修改DNTP混合物。此外，我们建议进一步探索该技术在应用中的实用性，例如单核苷酸多态性（SNP）检测和微阵列分析。总体而言，我们提出了一种新型热启动PCR的开发，该方法将为核酸扩增提供额外的特异性，并具有在许多基于PCR的平台中使用的灵活性。