Massarray designer

14 SNPs covering the whole TXNRD1 genetic variability were prioritized by a tagging approach, attempting to choose those most likely to be of functional relevance (nonsynonymous SNPs, SNPs located in the 5′ and 3′ UTR regions). SNPs associated with health status and longevity in Northern Europeans [14 (link), 15 (link)] were also chosen. SNPs with a minor allele frequency (MAF) less than 5% were excluded from the analysis.
Multiplex SNP genotyping was performed using iPLEX Gold Genotyping Assay and Sequenom MassARRAY (Sequenom, San Diego, CA, USA) according to manufacturer's instructions. Sequenom's MassARRAY Designer was used to design PCR and extension primers for each of the 14 SNPs selected. However, four of them (rs10861169, rs10861197, rs10047589, and rs4964287) were skipped by the software for primers design and were not analyzed in this paper. The details for genotyped SNPs are listed in Table 1.

Associations between the selected variants and breast cancer were evaluated in further studies. Three variants were genotyped in a set of 250 SBC patients and 248FNCCs. Genotyping of the variants c.339G>A and c.973G>A was performed with the MassARRAY platform (Sequenom, San Diego, CA, USA) using the iPLEX Gold Assay. The amplification and its extended primers were designed by MassARRAY Designer of Sequenom. The information on the primers was listed in Table 2. The amplification reaction conditions were as follows: an initial denaturation at 94°C for 15 min, followed by 45 cycles of denaturing at 94°C for 20 s, annealing at 56°C for 30 s, and the extension at 72°C for 60 s; finally, the reaction was elongated at 72°C for 3 min. Reaction parameters of single-base extension were an initial incubation at 94°C for 30 s, followed by 40 cycles at 94°C for 5 s with 5 nested cycles of 52°C for 5 s and 80°C for 5 s, respectively. Finally, singe-base extension was completed at 72°C for 3 min. Experimental data were analyzed by Typer software version 4.0 (Sequenom, San Diego, CA, USA). Genotyping of the variants c.51G>C and c.758C>A was done by PCR-sequencing assay. The primers and reaction conditions were the same as those used in the mutation screening for FANCC gene exon1 and exon7.