Shrimp Alkaline Phosphatase (SAP)

from deBacker et al. 2002

Shrimp Alkaline Phosphatase (SAP) has become one of today's most-selling DNA modifying enzymes due to the added convenience through a complete heat inactivation. While other alkaline phosphatases (from E. coli or Calf intestine) must be removed by extraction procedures, SAP is completely inactivated after 15 minutes at 65 °C. SAP works well in common buffers without the requirement for other additions.

Use in vector dephosphorylation:
Alkaline phosphatases are routinely used to reduce the background from empty, religated vectors during cloning of DNA fragments. SAP offers greater convenience to this procedure, since the enzyme may be completely removed by a simple heat inactivation step. In addition, SAP is active in all buffers used for restriction enzymes, so SAP can be added either during restriction digestion, or directly after. With SAP, the user can forget elaborate calculations and multi-step incubations, because the enzyme completely dephosphorylates DNA during one simple incubation.

Use in PCR cleanup[1]
USB Corporation has utilized SAP, in combination with Exonuclease I (ExoI) to offer the simplest, safest and most cost-effective method for removing nucleotides and primers from PCR products prior to sequencing or genotyping. In principle, SAP dephosphorylates nucleotides and ExoI degrades primers which interfere with the primer extension reaction. (Method of use covered by U.S Patent Numbers: 5,741,676; 5,756,285; 6,379,940 and 6,387,634)

Further convenience is added with the ExoSAP-IT reagent[2], (available from USB Corp. or GE Healthcare). With this reagent, the only hands-on procedure is a single pipetting step. After 15 min at 37 °C followed by 15 min at 80 °C (conveniently done in a thermocycler), the PCR product is ready for sequencing.

The procedure eliminates sample loss and handling errors, and is easily incorporated in robotic high-throughput systems.

Availability
Use of SAP for PCR cleanup requires purchase through a licensed supplier.  Since April 1^st 2003, USB Corporation has been the exclusive distributor of SAP in bulk quantities.

Specifications
Unit definition:  One Unit will convert 1 µmol of p-nitrophenyl phosphate per minute to nitrophenol and phosphate at 37ºC and pH 10.4 in 0.1 M glycine buffer, 1 mM each of ZnCl2 and MgCl2.

Specific activity: >2000 Units/mg

Purity: Purified to apparent homogeneity by SDS-PAGE. DNase, RNase or protease activities not detected.

Concentration: Minimum 10,000 Units/ml

Conversion to "Competitor Units"
Competitor products use different Unit definitions, and this makes a direct comparison on price difficult. We have tested SAP according to these unit definitions, and the results are shown below. In conclusion, SAP is always the most cost-effective alternative.

Properties:
Stability: Stable at -20 in storage buffer( 25 mM Tris-HCl pH 7.6, 1 mM MgCl2, 0.1 mM ZnCl2, glycerol 50 %). At 4 °C, 70 % activity remains after 15 days. At room temperature, 60 % activity remains after 96 hours. Completely inactivated after 15 minutes at 65 ºC. In a standard thermocycler, the process of heating from 37 to 95 and back to 37 °C is sufficient to completely inactivate SAP. 

Literature:

Olsen, R.L., Øverbø, K. and Myrnes, B. (1991): Alkaline phosphatase from the hepatopancreas of Shrimp (Pandalus borealis): A dimeric enzyme with catalytically active subunits. Comp. Biochem. Physiol. 99B: 755-761

de Backer, M., McSweeney, S., Rasmussen, H.B., Riise, B.W., Lindley, P. and Hough, E. (2002): The 1.9 Å crystal structure of heat-labile Shrimp Alkaline Phosphatase. J. Mol. Biol. 318: 1265-1274

[1] The use of SAP for PCR cleanup is covered by US Patents 5,741,676 and 5,756,285

[2] ExoSAP-IT is covered by US Patents 6,379,940 and 6,387,684