Pfuultra hf

A threonine-to-alanine point mutation at residue 153 (T153A) of human DARPP-32 was introduced by Pfu Ultra HF (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions and confirmed by sequencing. The sequences of the primers used were 5′-AGTCTGCTGG GCAAAAGGCA ACCTGTGGCC AGGGT-3′ (sense) and 5′-ACCCTGGCCA CAGGTTGCCT TTTGCCCAGC AGACT-3′ (antisense).

Point mutations (see Table 1) were introduced into the infectious molecular clone of wild-type H-1PV (pH1) [24 (link)] using the Quick Change Site-Directed Mutagenesis Kit (Qiagen, Hilden, Germany) to generate pH1-PMI, pH1-PMII, and pH1-PMIII. Specific PCR amplifications were performed in 50 µL reaction buffer containing 50 ng of pH1 template plasmid DNA, 125 ng of each forward and reverse primer (GATC Biotech, Konstanz, Germany), 200 µM of each dNTP, and 2.5 units of high fidelity DNA polymerase (PfuUltra HF, Agilent Technologies, Santa Clara, CA, USA). The PCR cycles were performed as following: 95 °C for 55 s for initial denaturation; 12 cycles of 95 °C for 30 s and 55 °C for 60 s for annealing; and 68 °C for 8 min for extension. To generate the double mutant, the PMI primers were used to introduce the PMI mutation into pH1-PMII. All constructs were verified by sequencing (GATC Biotech, Konstanz, Germany).