The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3'-terminal and 3'-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)-PCR and in real-time PCR with SYBR Green I dye and Taqman(R) probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.

中文翻译：

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使用可热激活引物的热启动 PCR：一种提高 PCR 性能的新方法。

聚合酶链反应 (PCR) 广泛用于需要高度特异性和可靠性的应用，例如基因检测、临床诊断、血液筛查、法医和生物防御。通过使用旨在防止 DNA 聚合酶延伸直至达到更严格、更高温度的热启动激活策略，已实现 PCR 性能的巨大改进。在此，我们提出了一种新的 PCR 热启动激活方法，其中引物在 3'-末端和 3'-倒数第二个核苷酸间连接处包含一个或两个不耐热的 4-氧代-1-戊基 (OXP) 磷酸三酯 (PTE) 修饰基团。研究表明，在 PCR 扩增之前存在的较低温度下，一种或多种 OXP PTE 修饰的存在会削弱 DNA 聚合酶引物的延伸。此外，发现在升高的温度下孵育 OXP 修饰的引物会产生相应的未修饰的磷酸二酯 (PDE) 引物，然后它是合适的 DNA 聚合酶底物。OXP 修饰的引物在具有终点检测的常规 PCR、一步逆转录 (RT)-PCR 和使用 SYBR Green I 染料和 Taqman(R) 探针检测的实时 PCR 中进行了测试。当在 PCR 中使用 OXP 修饰的引物替代未修饰的 PDE 引物时，观察到核酸靶标扩增的特异性和效率显着提高。OXP 修饰的引物在具有终点检测的常规 PCR、一步逆转录 (RT)-PCR 和使用 SYBR Green I 染料和 Taqman(R) 探针检测的实时 PCR 中进行了测试。当在 PCR 中使用 OXP 修饰的引物替代未修饰的 PDE 引物时，观察到核酸靶标扩增的特异性和效率显着提高。OXP 修饰的引物在具有终点检测的常规 PCR、一步逆转录 (RT)-PCR 和使用 SYBR Green I 染料和 Taqman(R) 探针检测的实时 PCR 中进行了测试。当在 PCR 中使用 OXP 修饰的引物替代未修饰的 PDE 引物时，观察到核酸靶标扩增的特异性和效率显着提高。