Author: Zhou, Yan; Zheng, HaiHong; Gao, Fei; Tian, DeBin; Yuan, ShiShan
Title: Mutational analysis of the SDD sequence motif of a PRRSV RNA-dependent RNA polymerase Document date: 2011_9_16
ID: yo9libo0_30
Snippet: To examine the function of the conserved SDD motif in PRRSV replication, a series of mutations were introduced into nsp9 by substituting each of the three residues (positions 3050, 3051, and 3052) (Figure 1 ). PCR-mediated mutagenesis was carried out with primers containing the required nucleotide changes ( Table 2) . To construct the other mutations, such as pSND, SOE PCR was used with pAP-RRS DNA as the template and two pairs of primers, SF8129.....
Document: To examine the function of the conserved SDD motif in PRRSV replication, a series of mutations were introduced into nsp9 by substituting each of the three residues (positions 3050, 3051, and 3052) (Figure 1 ). PCR-mediated mutagenesis was carried out with primers containing the required nucleotide changes ( Table 2) . To construct the other mutations, such as pSND, SOE PCR was used with pAP-RRS DNA as the template and two pairs of primers, SF8129 plus SR9348SND and SF9301SND plus SR10211 ( Table 2 ). The PCR product containing the mutation and pAPRRS was digested by Avr II and EcoR V, used to replace the Avr II-EcoR V fragment (8958-12270 nt) of pAPRRS. The intro-duced mutations were verified by sequencing and restriction analysis. Mutant plasmids were transfected into BHK-21 cells, whose actual infectivity was examined.
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