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 KRAS 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.