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.
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| Fig. 1. Summary of ExoSAP-IT PCR product treatment. |
ExoSAP-IT utilizes two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, 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. Shrimp Alkaline Phosphatase removes the remaining dNTPs from the PCR mixture.
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| Fig. 4. ExoSAP-IT treatment of primers. A 5' FAM-labeled 20-mer was electrophoresed on a 15% denaturing polyacrylamide gel before (pre) and following (post) ExoSAP-IT treatment (0.1 pmol per lane). The oligo and subsequent digestion products were visualized on a fluorescent scanner. ExoSAP-IT is able to digest at least 25 pmol of primers (in 5 µl) in 15 min at 37°C, which is about 10 times the average concentration of primers that are present in a typical PCR reaction. The digestion product is a 5' FAM-labeled dinucleotide. Fluorescein-labeled dCTP is shown as a marker. |
Rapid PCR Product Clean-Up 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.
Simple: Single-Step
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 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).
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| 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 may be used as an effective clean-up method prior to fluorescent or radioactive DNA sequencing (Fig. 3), SNP analysis or any other application requiring a PCR product free of excess nucleotides and primers.
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| Fig. 3. Fluorescent sequencing results of a 100 bp pUC18 PCR fragment sequenced with a -20 Fwd primer using fluorescent sequencing reagents. PCR clean-up performed with: (a) ExoSAP-IT; (b) a column designed for PCR clean-up. Base miscalls in (b) are due to inherently low yields of short PCR products when using columns. |
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