Shrimp alkaline phosphatase

To minimize the misincorporation of different nucleotides during PCR reaction, PCR amplification is performed using LA Taq polymerase (Takara) since this has relatively high fidelity, and the number of PCR cycles is kept to 20. Excess primers and dNTPs are treated with shrimp alkaline phosphatase and Exonuclease I. 

Shrimp alkaline phosphatase (SAP) (United States Biochemical, Cleveland, OH). This enzyme is preferred to calf intestinal phosphatase (CIP) because it is heat-labile in its 10X reaction buffer 200 mM Tris-HCl, pH 8.0,100 mMMgCl2. 

Shrimp alkaline phosphatase (1 unit/pL, New England Biolabs or other suitable vendor). 

Remove dNTPs and oligonucleotide primers from the PCR product by combining 20 pL of PCR product with 2 pL exonuclease I and 2 pL shrimp alkaline phosphatase (SAP). Incubate at 37°C for 30min followed by 15 min at 85°C. 

IX Shrimp Alkaline Phosphatase Buffer (Roche Diagnostics Coip., Indianapolis, IN, cat. no. 1 758 250) or equivalent. Store at -20°C. 

U of Shrimp Alkaline Phosphatase, which removes excess dNTPs from the PCR... 

The original PROBE assay has been replaced now by a bead-free, homogeneous assay called the MassExtend (hME) assay. Before the primer extension reaction, shrimp alkaline phosphatase (SAP) is added to the PCR product. This dephosphorylates any residual deoxynucleotides that otherwise would inter-... 

As the third step of the GOOD assay is a primer extension with a specifically tailored set of a-S-dNTPs and a-S-ddNTPs, dNTPs of the PCR have to be removed. This is done enzymatically by degradation of the dNTPs in the second step by shrimp alkaline phosphatase (SAP). 

Figure 2. Flowchart of the GOOD assay. SAP stands for shrimp alkaline phosphatase digestion, PE for primer extension, PDEfor phosphodiesterase. Alkyl for alkylation and MS...

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