Comparison of purification protocols for effective large-primer removal between rounds of PCR amplification

Effective polymerase chain reaction (PCR) product purification is essential for downstream applications like next-generation sequencing (NGS). While standard protocols remove short primers (20–30 nucleotides [nt]), NGS workflows require efficient removal of larger primers (40–50 nt) to prevent amplification artifacts. This study compared commercial kits (magnetic beads, silica columns, enzymatic degradation) with traditional isopropanol/ethanol precipitation and simple dilution for their ability to remove large primers from a 161-bp <i>KRAS</i> PCR product. Efficacy was assessed by a secondary PCR designed to amplify remaining primers. Optimal yield was achieved with isopropanol precipitation with NH₄Ac (2.5–3.0 M) and overnight incubation at 4 to −20 °C. Interestingly, a simple 1:200 dilution showed comparable results. Among commercial kits, magnetic beads demonstrated superior primer removal, as evidenced by a substantially lower concentration of the secondary PCR product (0.3 ng/µL ± 0.23) compared to untreated samples (22.13 ng/µL ± 1.7). For NGS workflows, magnetic beads are the most effective method for removing large primers, while isopropanol precipitation and dilution offer viable, low-cost options, particularly for workflows involving multiple PCR steps. Efficient PCR product purification is critical for downstream applications like next-generation sequencing (NGS), yet standard methods often fail to remove large primers, causing sequencing artifacts. We compared the effectiveness of magnetic beads, silica columns, enzymatic degradation, isopropanol precipitation, and simple dilution for removing these problematic primers. While magnetic beads offered the most complete purification, traditional isopropanol precipitation also performed well as a cost-effective method. Notably, a simple 1:200 sample dilution emerged as a surprisingly effective and highly economical alternative. This work provides researchers with validated and optimized cleanup strategies, enhancing data reliability for workflows that depend on high-fidelity DNA, particularly those involving multiple amplification steps. This study evaluated several post-PCR primer removal methods for multi-step amplification workflows. We present an optimized isopropanol precipitation protocol and a simple 1:200 dilution as efficient and cost-effective alternatives to the magnetic bead purification standard used in NGS. We present an optimized isopropanol precipitation protocol that effectively removes large primers between PCR steps.The study validates low-cost strategies for a critical step in multi-round PCR workflows.Simple sample dilution can serve as a surprisingly effective, ultra-low-cost cleanup method.Our data challenge the assumption that expensive methods are required for multi-step PCR workflows. We present an optimized isopropanol precipitation protocol that effectively removes large primers between PCR steps. The study validates low-cost strategies for a critical step in multi-round PCR workflows. Simple sample dilution can serve as a surprisingly effective, ultra-low-cost cleanup method. Our data challenge the assumption that expensive methods are required for multi-step PCR workflows.

高效的聚合酶链式反应（PCR）产物纯化，是二代测序（NGS）等下游实验的核心前提。尽管常规纯化方案可去除20~30个核苷酸（nt）的短引物，但二代测序（NGS）流程需要高效去除40~50 nt的长引物，以避免扩增伪影的产生。
本研究以161 bp的KRAS基因PCR产物为模板，对比了商业化纯化试剂盒（磁珠法（magnetic beads）、硅胶柱法（silica columns）、酶降解法（enzymatic degradation））、传统异丙醇/乙醇沉淀法（isopropanol/ethanol precipitation）与简单稀释法（simple dilution）去除长引物的效果。纯化效果通过靶向扩增残留引物的二次PCR进行评估。
采用含2.5~3.0 M乙酸铵（NH₄Ac）的异丙醇沉淀法，并于4℃至-20℃孵育过夜，可获得最优的产物回收率。值得注意的是，采用1:200比例的简单稀释法，也可达到相近的纯化效果。在商业化试剂盒中，磁珠法展现出最优的引物去除效果：未处理样品的二次PCR产物浓度为22.13 ng/μL ±1.7，而磁珠法处理后的产物浓度仅为0.3 ng/μL ±0.23，差异显著。
针对二代测序（NGS）流程，磁珠法是去除长引物的最优方案；而异丙醇沉淀法与稀释法则是成本低廉且可行的替代方案，尤其适用于多轮PCR扩增流程。
高效的PCR产物纯化对二代测序（NGS）等下游应用至关重要，但常规方法往往无法去除长引物，进而引发测序伪影。本研究对比了磁珠法、硅胶柱法、酶降解法、异丙醇/乙醇沉淀法与简单稀释法去除此类问题引物的效果。
磁珠法可实现最彻底的引物去除，而传统异丙醇/乙醇沉淀法同样表现优异且成本低廉；尤为值得关注的是，1:200比例的简单样本稀释法，是一种效果出众且成本极低的替代方案。本研究为研究者提供了经过验证与优化的PCR产物纯化策略，可提升依赖高保真DNA的实验流程的数据可靠性，尤其是涉及多轮扩增的实验。
本研究针对多轮扩增流程，评估了多种PCR后引物去除方案；并提出优化后的异丙醇沉淀法与1:200简单稀释法，可作为二代测序（NGS）流程中磁珠纯化法的高效低成本替代方案。本研究证实，优化后的异丙醇沉淀法可在PCR扩增步骤间有效去除长引物，同时验证了多轮PCR流程中关键步骤的低成本纯化方案。简单样本稀释法是一种效果出众、成本极低的纯化手段。本研究的数据推翻了“多步PCR流程必须使用昂贵纯化方法”的固有认知。