PMC:1079843 / 13985-15891 JSONTXT

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The other multiplex PCR was performed under the same conditions, but using 0.8 μl of primer mix containing 0.1 μM of the primer pairs for 5'flankingSNP1, 5'flankingSNP2, 3'UTRSNP1, and 3'UTRSNP2. A 4 μl aliquot of the PCR products was treated with 5 U of shrimp alkaline phosphatase (SAP) and 0.1 U of exonuclease I in a total volume of 10 μl to remove primers and unincorporated dNTPs; the reaction was carried out at 37°C for 1 hour, then terminated by incubation at 72°C for 15 minutes to inactivate the enzymes. The multiplex SBE reaction was carried out using SNP-specific primers and fluorescent-labeled terminators (the ABI PRISM SNaPshot Multiplex Kit). The short-extension reaction was performed on a thermal cycling machine for 25 cycles of 10 s at 96°C, 5 s at 50°C, and 30 s at 60°C. After short-extension, excess ddNTPs were removed from the SBE products by addition of 0.5 U of SAP to the reaction mixture and incubation at 37°C for 1 hour. 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