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在PCR放大轮次之间有效去除大型原料的净化方案的比较

Eleni Polatoglou¹, Abel Bronkhorst¹, Victor Costina²

¹Munich Biomarker Research Center, Institute of Laboratory Medicine, TUM University Hospital, German Heart Center Munich, Munich, Germany.

|December 13, 2025

概括

对聚合酶连锁反应 (PCR) 产品的有效净化对于下一代测序 (NGS) 至关重要. 磁珠在去除NGS中的大型原料中最有效,而异醇沉和稀释提供了具有成本效益的替代方案.

科学领域:

分子生物学分子生物学
生物技术是生物技术.

背景情况:

聚合酶链反应 (PCR) 产品净化对于下游应用至关重要.
下一代测序 (NGS) 工作流需要高效地去除较大的启动器 (40-50 nt),以避免放大工件,而不是针对较短的启动器 (20-30 nt) 的标准协议.

研究的目的:

为了比较各种方法的有效性,从PCR产品中去除大型原料.
确定NGS工作流程的最佳净化策略.

主要方法:

商业套件 (磁珠,柱,酶降解) 与传统的异醇/乙醇沉和简单稀释的比较.
评估使用二次PCR来放大残留原料的原料去除功效.
优化异醇沉条件 (试剂度,化温度和时间).

主要成果:

用氨酸 (NH4Ac) 沉异醇和一夜间化产生了最佳的结果.
一个简单的1:200稀释证明了与沉方法相比较的原料去除.
磁珠在商业套件中表现出优异的性能,与未经处理的样本相比,显著降低了二次PCR产品度 (0.3 ng/μL vs 22.13 ng/μL).

结论:

磁珠是NGS工作流程中大型原料去除的最有效方法.
异醇沉和稀释提供了可行的,低成本的替代方案,特别是在多步PCR过程中.

关键词:

清理NGS的清理工作在PCR净化过程中.大型打印机的移除方式通过多次PCR进行PCR.拆除原料拆除原料拆除

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