Lightsnip

DNA isolation was performed by QIAamp DNA Blood Mini Kit (Cat. No. 51304, QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Genotypes of VDBP rs2298850, rs4588, and rs7041 SNPs were determined by LightSNiP assay using simple probes (LightSNiP, TibMolBiol, Berlin, Germany) and LightCycler Fast Start DNA Master HybProbe Kit (Cat. No.12239272001, Roche Diagnostics, Mannheim, Germany). Real-time PCR (RT-PCR) was performed with LightCycler 480 Instrument II (Roche Diagnostics, Mannheim, Germany). Genotyping was performed as previously explained [23 (link)].

DNA was extracted from LCLs using the conventional salting-out procedure by the MasterPure complete DNA and RNA purification kit (Epicentre, an Illumina company, CA, USA). Ten common polymorphisms (MAF ≥ 25%) in nine genes involved in folate uptake and metabolism were genotyped by means of TaqMan (Applied Biosystems, Foster City, CA, USA) or LightSNiP (TIB MOLBIOL, Germany) probes, in accordance with the manufacturers’ instructions. Genotyping of rs1051266 (SLC19A1) was done using LightSNiP, while genotyping of the following polymorphisms was performed using TaqMan probes: rs1544105 (FPGS), rs1677693 (DHFR), rs2236225 (MTHFD1), rs1801133 and rs1801131 (MTHFR), rs1801394 (MTRR), rs10948059 (GNMT), rs2424913 (DNMT3B), and rs3733890 (BHMT).

DNA isolation was performed by QIAamp DNA Blood Mini Kit (Cat. No. 51304, QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Genotypes of VDBP rs2298850, rs4588 and rs7041 SNPs were determined by LightSNiP assay using simple probes (LightSNiP, TibMolBiol, Berlin, Germany) and LightCycler Fast Start DNA Master HybProbe Kit (Cat. No.12239272001, Roche Diagnostics, Mannheim, Germany). Real-time PCR (RT-PCR) was performed with LightCycler 480 Instrument II (Roche Diagnostics, Mannheim, Germany), and genotyping was done by using melting curve analysis as previously described [31 (link)].

The DNA was extracted from buccal swabs using QIAamp DNA mini kits (Qiagen) or MasterPure complete DNA and RNA purification kits (Epicentre (Illumina) Madison, WI, USA), according to the manufacturer instructions.
The interactions between the genes of interest were evaluated by text and database mining using STRING 10.0 [18 (link)]. For each of the selected genes, at least one polymorphism was chosen for the genotype analysis, which showed a minor allele frequency ≥25% and the highest number of PubMed connections to CHD and/or etiologically related conditions (e.g., NTD and orofacial cleft). Ten common polymorphisms in nine genes involved in the folate and methionine cycles were analyzed using the TaqMan (Applied Biosystems, Foster City, CA, USA) or LightSNiP (TIB MOLBIOL, Berlin, Germany) probes, according to the manufacturer instructions. The following polymorphisms were genotyped using TaqMan probes: rs1544105 (FPGS) (assay number C_8342611_10), rs1677693 (DHFR) (assay number C_3103231_10), rs1801133 and rs1801131 (MTHFR) (assay numbers C_1202883_20 and C_850486_20), rs1801394 (MTRR) (assay number C_3068176_10), rs2236225 (MTHFD1) (assay number C_1376137_10), rs3733890 (BHMT) (assay number C_11646606_20), rs10948059 (GNMT) (assay number C_11425842_10), and rs2424913 (DNMT3B) (assay number C_25620192_20). Genotyping of rs1051266 (SLC19A1) was carried out by LightSNiP.

DNA was extracted from samples of peripheral blood taken on EDTA using Maxwell 16 blood DNA purification kit (Promega Corp., Madison, WI, USA) or silica membranes (Qiagen, Hilden, Germany), following the recommendations of the manufacturers. BSG and SLC16A1 polymorphic variants were determined using the Taqman (Thermo Fisher, Waltham, MA, USA) and LightSNiP (TIB MOLBIOL, Berlin, Germany) assays. PCR was performed on a LightCycler 480 II device (Roche Diagnostics, Rotkreuz, Switzerland), according to the manufacturer’s recommendations.

We extracted genomic DNA from 200 µL EDTA blood with the help of the QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The different polymorphisms were determined by melting curve analysis. We used LightSNiP (SimpleProbe) assays from TIB-MolBiol (Berlin, Germany). In total, 1 µL of sample DNA was added to 0.5 µL of the LightSNiP reagent mix, 5 µL of Blue ProbeqPCR 2× Mix (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) and 3.5 µL PCR grade water (Invitrogen, Paisley, UK). We performed real-time PCR on a LightCycler^® 96 system (Roche, Germany) as indicated by the protocol of the manufacturer.