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Direct sequencing of PCR products is a rapid and convenient method of sequence analysis compared to cloning PCR fragments first. Several key factors are important when preparing PCR products for direct sequencing. Verification of the desired product is essential, which includes confirming a lack of non-specific products and primer dimers. Clean-up of the reaction mixture is also necessary to remove unincorporated primers and dNTPs that can interfere with subsequent reactions and lead to an unreadable sequence.
Classical methods used to clean up PCR products prior to sequencing include gel electrophoresis, ethanol precipitation, and column chromatography. While quite effective, these methods can quickly become cumbersome when processing multiple samples in parallel and exhibit varying degrees of sample loss. The ideal method of PCR product preparation prior to sequencing should have zero sample loss, be simple to execute, and provide consistent results. USB Corporation has addressed these issues by developing ExoSAP-IT® for PCR product clean-up. ExoSAP-IT® is an effective alternative to classical methods of PCR product clean-up prior to sequencing.
Novel Method for PCR Clean-UpExoSAP-IT® is unique in that it is a one-step enzymatic treatment of PCR products to eliminate unincorporated primers and dNTPs so that they cannot interfere with downstream sequencing reactions. ExoSAP-IT®, based on Exonuclease I and Shrimp Alkaline Phosphatase, is added directly to the PCR product to degrade primers and dephosphorylate dNTPs that were not consumed in the reaction. Treatment is carried out for 15 minutes at 37°C, followed by a 15 minute incubation at 80°C to completely inactivate both enzymes. PCR products are then immediately ready for downstream sequencing reactions without any additional manipulation.
This novel enzymatic treatment of PCR products prior to sequencing reliably removes primers from the mixture and exhibits zero sample loss (Fig. 1), in contrast to the sample loss observed with other methods. Low recovery of small PCR products (e.g., 100 bp) with column-based methods can lead to base miscalls in sequencing data, while sequencing ExoSAP-IT®-treated products yields the correct sequence (Fig. 2).
Conclusion
Compared to classical methods of PCR product purification prior to sequencing, ExoSAP-IT® makes it easier to process multiple samples in parallel. Only one pipetting step is required to treat a PCR product with ExoSAP-IT®, compared to multiple steps required for ethanol precipitation, gel electrophoresis, or column methods. The simplicity of ExoSAP-IT® and scalability makes this method a practical way to easily process samples either manually or in high-throughput applications.
For further information, contact:
USB Corporation
E-mail: techsupport@usbweb.com
Phone: 800.321.9322/216.765.5000
