Genotyping of the AZD2281 order p.F362V variant in 80 Iranian Jewish controls and the non-exome-sequenced family members (Figure 1; family

A: I.1, I.2, II.2, II.3, and II.4 and family B: I.1, I.2, II.1) was performed at the Gertner Institute of Human Genetics, Sheba Medical Center, Israel. Sanger Sequencing (Figure 1B) or restriction digest with the restriction enzyme Alw26I (data not shown) were used to perform this genotyping. Both methods used the following custom primer sequences: forward: 5′-CTTTCAATTATTTCCAAAAATCAAATC-3′ and reverse: 5′-CACTGTCATACTGAAAGATGATAGAAA-3′. These primers resulted in a 286 bp amplicon that targeted the nucleotide of interest. The p.F362V variant, found in families A and B, was validated in these three samples using all three methods: TaqMan genotyping, Sanger sequencing, and restriction digestion. Sanger sequencing http://www.selleckchem.com/products/BMS-777607.html of PCR-amplified products was used to genotype p.R550C and p.A6E variants. The following custom primers were used for p.A6E: forward: 5′-GCCGGTTGAATGTAGAGGTC-3′ and reverse: 5′-CCAAAGCAGCAGTTGGTGTA-3′. The following custom primers were used for p.R550C: forward: 5′-GCCATTTTAAGCCATTTTGC-3′ and reverse: 5′-TTTCCCTTTTCCTAGCTTACCC-3′. The mutations p.R550C and p.A6E were genotyped in 300 French Canadian healthy controls. In addition, p.R550C was genotyped in 225 Bangladeshi healthy controls. Full-length cDNA

encoding human ASNS was amplified from first-strand cDNA derived from the HEK293 human kidney cell line with an RNeasy plus mini kit (QIAGEN), High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), Phusion HF DNA polymerase (Finnzymes), and a specific primer set (5′-CTCGAGATGTGTGGCATTTGGGCGCT-3′ and 5′-CTCGAGCCTAAGCTTTGACAGCTGACT-3′). The cDNA was subcloned into the pCR-Blunt II-TOPO vector (Invitrogen-Life Technologies) and subjected to sequence Levetiracetam analysis (pCR-Blunt II-ASNS-WT). Using pCR-Blunt II-ASNS-WT, A6E, F362V, and R550C of ASNS were made by PCR-mediated site-directed mutagenesis using Phusion HF DNA polymerase and a specific primer set (A6E: 5′-GCTGTTTGGCAGTGATGATTG-3′ and 5′-TCCCAAATGCCACACATCTC-3′; F362V: 5′-GTCTCTGGAGAAGGATCAGA-3′ and 5′-GATCACCACGCTATCTGTGT-3′; R550C:

5′-GCACGCTGACCCACTAC-3′ and 5′-AGGCAGAAGGGTCAGTGC-3′), which were phosphorylated by T4 polynucleotide kinase (New England BioLabs). The amplicons were self-ligated using T4 DNA ligase (Promega) and subjected to sequence analysis (pCR-Blunt II-ASNS-A6E, pCR-Blunt II-ASNS-F362V, and pCR-Blunt II-ASNS-R550C). ASNS human cDNA containing each allele was subcloned into the pcDNA3.1(+) vector (Invitrogen-Life Technologies) using the KpnI and XbaI sites from pCR-Blunt II-ASNS-WT, pCR-Blunt II-ASNS-A6E, pCR-Blunt II-ASNS-F362V, or pCR-Blunt II-ASNS-R550C and subjected to sequence analysis (pcDNA3.1(+)-ASNS-WT, pcDNA3.1(+)-ASNS-A6E, pcDNA3.1(+)-ASNS-F362V, or pcDNA3.1(+)-ASNS-R550C; Figure S2). Using pcDNA3.1(+)-ASNS-WT, pcDNA3.1(+)-ASNS-A6E, pcDNA3.