Pfu ultra dna polymerase system

The DENV2 16681 C-prM-E expression construct (Ansarah-Sobrinho et al., 2008 (link)) was used as a template for site-directed mutagenesis using the Pfu Ultra DNA polymerase system (Cat# 600380; Agilent Technologies, Santa Clara, CA) and primers generated by QuikChange Primer Design (Agilent Technologies). The entire C-prM-E region was sequenced (Quintara, San Francisco, CA) to confirm the presence of the desired mutation(s).

We used a previously described expression vector encoding the structural genes (C-prM-E) of the WNV NY99 strain [57 (link)] as a template for mutagenesis. Initially, threonine at residue 198 of the WNV E protein was replaced with phenylalanine by site-directed mutagenesis using the Pfu Ultra DNA polymerase system (Agilent Technologies). The reciprocal mutation (F193T) was introduced into a previously described expression vector encoding the structural genes (C-prM-E) of the DENV1 Western Pacific strain [86 (link)]. Mutation at the analogous residue of the ZIKV E protein (F198T) was introduced into a plasmid encoding the structural genes of the ZIKV strain H/PF/2013 [120 ]. This plasmid is described elsewhere [121 ]. Additional amino acid variants were introduced at position 198 of the WNV E protein using primers containing a degenerate codon (NNN). PCR cycling parameters were as follows: 1 cycle of 95°C for 1 min; 18 cycles of 95°C for 50 s, 60°C for 50 s, and 68°C for 9 min; and 1 cycle of 68°C for 7 min. PCR products were treated with DpnI (New England BioLabs) for 3 h at 37°C, prior to transformation into Stbl2 cells (Invitrogen) and propagation at 30°C. The entire C-prM-E region of each construct was sequenced to ensure that no additional mutations were present.