ExoSAP-IT® For PCR Product Cleanup
Single-step enzymatic cleanup of PCR products that eliminates unincorporated primers and dNTPs
- Conserves PCR samples — 100% recovery of both short and long PCR products
- One tube/one step PCR cleanup — Add ExoSAP-IT reagent directly to PCR product
- Eliminates spin columns — Decreases time and expense while increasing yield
- Removes contaminating primers and dNTPs — No interference in downstream applications
- Scalable — Economical for high-throughput purification
- Simple processing — Robotic-friendly; Replaces beads, filtrations, and plates
- Generates less waste than columns
ExoSAP-IT reagent is designed for simple, quick PCR cleanup for downstream applications, such as DNA sequencing or Single Nucleotide Polymorphism (SNP) analysis. When PCR amplification is complete, any unconsumed dNTPs and primers remaining in the PCR product mixture will interfere with these methods. ExoSAP-IT removes these contaminants.
ExoSAP-IT is added directly to the PCR product and incubated at 37°C for 15 minutes (Fig. 1). After PCR treatment, ExoSAP-IT is inactivated simply by heating to 80°C for 15 minutes.
|Fig. 1. Summary of ExoSAP-IT PCR product treatment.|
ExoSAP-IT single-step PCR cleanup utilizes two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase (SAP), together in a specially formulated buffer, to remove unwanted dNTPs and primers from PCR products. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced in the PCR. SAP removes the remaining dNTPs from the PCR mixture.
Rapid PCR Product Cleanup Protocol
ExoSAP-IT requires only one pipetting step and two incubations. Just add ExoSAP-IT to the PCR product and within 30 minutes sequencing or SNP analysis can be performed.
The method is designed to require a minimum of ‘hands-on’ time. Enzymatic removal of excess primers and unincorporated nucleotides occurs in one easy step by using ExoSAP-IT reagent in a single tube or microtiter well. Only simple pipette transfers are required, therefore, many samples can be processed at once, either manually or with robotics.
No Sample Loss
Use of ExoSAP-IT reagent eliminates all gel or column purifications, sedimentations, filtrations, beads, and/or magnetic separations(1). There is 100% recovery of both short and long PCR products with ExoSAP-IT (Fig. 2).
|Fig. 2. ExoSAP-IT treatment of PCR products with no sample loss. Single-copy targets were amplified from human genomic DNA. HES-1 (125 bp), numb (455 bp), NRAGE (1.55 kb), and numb (4.6 kb) were loaded on a 1.5% agarose gel before (pre) and after (post) ExoSAP-IT treatment. M is the DNA marker lane. Note that a variety of PCR product sizes can be treated with ExoSAP-IT, even a 125 bp fragment, with no sample loss.|
Achieve High Data Quality from PCR Products
ExoSAP-IT reagent may be used as an effective cleanup method prior to fluorescent or radioactive DNA sequencing, SNP analysis, or any other application requiring a PCR product free of excess nucleotides and primers.
Fig. 1. Fluorescent sequencing results of a 1,007 bp PCR product. A 1,007 bp fragment was amplified and treated with ExoSAP-IT reagent (above) or an alternate enzymatic reagent (below) and sequenced. Pherograms revealed no miscalls with ExoSAP-IT reagent but four miscalls at position 25, 26, 30, and 39 with the alternate enzymatic reagent. *5 µl of PCR product was treated with 2 µl ExoSAP-IT reagent or the alternate enzymatic method per its protocol. Note: Two of the four miscalls are frame-shifts.
Fig. 2. Fluorescent sequencing results of a 100 bp pUC18 PCR fragment sequenced with a -20 Fwd primer using fluorescent sequencing reagents. PCR cleanup performed with: (a) ExoSAP-IT reagent; (b) a column designed for PCR cleanup. Base miscalls in (b) are due to inherently low yields of short PCR products when using columns. *5 µl of PCR product was treated with 2µl ExoSAP-IT reagent or 1µl Exo I and 1µl Alkaline phosphatase from an alternative enzymatic method using the recommended incubation conditions (15 minutes at 37°C followed by 15 minutes at 80°C) in 16 duplicates each. The treated PCR products were then sent for automated sequencing. Sequencing results over the first 600 bp consistently show more miscalls with the samples treated with the alternative enzymatic method than samples treated with ExoSAP-IT reagent. The pherogram shown here is an example. Product sequenced after using the alternative enzymatic method showed 3 miscalls and a 1 base frame shift. The same sequence after ExoSAP-IT reagent treatment was completely accurate with no miscalls or errors.
- DUGAN, K. A., LAWRENCE, H. S., HARES, D. R., FISHER, C. L. AND BUDOWLE B. (2002) J. Forensic Sci 47, 811-818.
- HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.
- MU, J., DUAN, J., MAKOVA, K., JOY, D., HUYNH, C., BRANCH, O., LI, W. AND SU, X. (2002) Nature 418, 323-326.
- SILVA, JR., W. A., COSTA, M. C. R., VALENTE, V., DE FREITAS SOUSA, J., PACÓ-LARSON, M. L., ESPREAFICO, E. M., CAMARGO, S. S., MONTEIRO, E., DE JESUS, A., HOLANDA, M. A., ZAGO, M. A., SIMPSON, A. J. G. AND NETO, E. D. (2001) BioTechniques 30, 537-542.
- WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
Shipped on dry ice. Store at -20°C.