PCR product cleaning before Sanger sequencing
PCR products are the most common template for Sanger sequencing to confirm the right sequence or detect a single or a few mutations. To achieve high-quality sequencing results, the PCR product must be a single product, a single band judged by agarose gels, and shall be purified to remove the remaining primers and dNTPS. Although there are cases that unpurified PCR products may generate good Sanger sequencing traces (probably because the concentration of remaining primers and dNTPs is low, not enough to cause problems?), components in unpurified PCR products could interfere with the downstream cycle sequencing reactions and lead to high background. To eliminate the risk, most operators choose to purify the PCR products before proceeding to Sanger sequencing.
Methods for purification of PCR products
There are multiple ways to remove the interference from the remaining primers and dNTPs, including ethanol precipitation, agarose gel extraction, spin columns, magnetic beads, and enzymatic digestions. Ethanol precipitation is time-consuming, causes sample loss, and is less common now. Agarose gel extraction is still available, but it is preferably used when a single PCR product needs to be separated from other contaminating products on agarose gels. However, since agarose may contain impurities that interfere with the downstream cycle sequencing reaction, optimizing the PCR conditions, if possible, to generate a single PCR product for sequencing is highly recommended. Spin columns and magnetic beads bind the PCR products, and the short primers and small dNTP molecules are washed away. Then, the PCR products are eluted from the spin columns or beads. These are efficient methods and takes only 20 minutes or so, but the recovery rate for PCR products is reported to be 60 to 90% depending on the length of the PCR products and the suppliers. The enzymatic digestion method is easy and needs little hands-on time compared with other methods. Because the enzymatic reaction happens in a single tube for digestion and enzyme inactivation, the method is highly valued and widely used in PCR template preparation for Sanger sequencing.
ADS PCR Cleaning Beads for PCR product Purification
ADS PCR Cleaning Beads are magnetic beads optimized for purification of PCR products. With the cleaning beads added directly to the PCR product reactions, the PCR products are selected bound and eluted, away from primers and dNTPs. The ADS Cleaning Beads can release the DNA with high recovery rate as high as 90% and the eluted, purified PCR products are directly quantified and evaluated for quality and purity using a Nanodrop for instance. Read our application notes to learn more about this cost-effective PCR purification product.
ADS Exo-Alp PCR Cleanup Mix for Enzymatic Digestions
Enzymatic digestion methods mentioned above use an alkaline phosphatase to dephosphorylate dNTPs and an exonuclease to cleave single strand primers into single nucleotides without degrading double strand PCR products. The dephosphorylated dNTPs and the single nucleotides would not interfere the downstream cycle sequencing reactions. Exo-Alp PCR Cleanup Mix made by AdvancedSeq is a high-quality alternative to ExoSAP-IT PCR Product Cleanup Reagent with comparable performance. The cost-effective feature allows the on-budget labs to save money without compromising the quality. You can find performance comparison in our application note.
If you are interested in exploring these products or other Sanger sequencing reagents, contact us or visit our website.