Iplex enzyme

We selected SNPs (rs8051542, rs12443621, rs3803662, rs4784227 and rs3112612) in the TOX3/LOC643714 locus at 16q12.1 from the confirmatory results from GWAS or meta-analysis in multiple populations [6 (link),7 (link),11 (link),20 (link)].
The five SNPs were genotyped using the SEQUENOM MassARRAY matrix-assisted laser desorption ionization-time of flight mass spectrometry platform (Sequenom, San Diego, CA, USA). Primers for multiplex PCR and extended reactions were designed using proprietary software (Assay Designer, version 3.1) provided by Sequenom Inc (San Diego, CA, USA). In accordance with the manufacturer’s instructions, SNPs were genotyped using Sequenom MassARRAY genotyping technology (Sequenom, San Diego, CA, USA) and amplified in multiplex PCR by a standard PCR protocol. The genomic amplification product was cleaned using shrimp alkaline phosphatase (Sequenom, San Diego, CA, USA) to neutralize any unincorporated dNTPs, followed by a single-base extension reaction using the iPLEX enzyme (Sequenom, San Diego, CA, USA) and mass-modified terminators (Sequenom, San Diego, CA, USA). The products of the iPLEX reaction were desalted and transferred onto a SpectroCHIP (Sequenom, San Diego, CA, USA) by the MassARRAY nanodispenser (Sequenom, San Diego, CA, USA), which was then analyzed by the MassARRAY analyzer by combining base calling with the clustering algorithm.

All procedures were performed as previously described [13] . Briefly, DNA was isolated from peripheral blood mononuclear cells by the QIAamp DNA Mini-Kit (QIAGEN, Hilden, Germany). Genotyping of BDNF rs6265 single nucleotide polymorphism (SNP) was conducted with the MassARRAY platform (SEQUENOM, San Diego, CA) as described elsewhere [17] . PCR-primers were generated with the Assay Designer 4.0 software (SEQUENOM). Multiplex PCR reactions were performed with 12.5 ng of genomic DNA, 500 µM dNTPs (ABgene, Hamburg, Germany), 100 nM PCR primers, 1.625 mM MgCl2 and 0.5 U HotStar Taq polymerase (QIAGEN). Shrimp alkaline phosphatase (SAP) treatment, an iPLEX reaction cocktail with extension primers (7–14 µM), a iPLEX termination mix and an iPLEX enzyme (SEQUENOM) were added to the PCR-products. The resulting extension products were desalted using SpectroCLEAN resin (SEQUENOM), then spotted on SpectroCHIPs GenII (SEQUENOM) and analyzed with the MassARRAY MALDI-TOF mass spectrometer. Typer 3.4 Software was used to identify allele specific extension products and resulting genotypes (SEQUENOM). For genotyping quality assurance CEU HapMap Trios (Coriell Institute for Medical research, Camden, NJ) were included and compared with the HapMap-CEU population (www.hapmap.org). For all analyses val/val homozygotes ( = GG-carriers) were compared against met-carriers (AG- and AA-carriers).