Genotyping master mix

Manufactured by Thermo Fisher Scientific

Sourced in United States, Austria

Genotyping Master Mix is a pre-formulated reagent solution designed for efficient and reliable genotyping assays. The product contains all the necessary components, including DNA polymerase, buffer, and optimized reagents, for performing genotyping experiments.

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40 protocols using genotyping master mix

Genotyping Putative Deleterious Variants

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An ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and TaqMan assays with custom probes were used to genotype putative deleterious variants. Custom probe sequences are available in S2 Table. Each 384-microtiter plate contained two non-template controls and two samples with the variant. The reactions and data analysis were performed using Genotyping Master Mix and Sequence Detection Systems, respectively, according to the standard protocols (Applied Biosystems, Foster City, CA, USA). Differences in genotype distribution between cases and controls were tested with one-sided, Fisher’s exact tests.
We computed the effective sample size and statistical power using a web browser program, Genetic Power Calculator developed by Purcell et al. (http://pngu.mgh.harvard.edu/~purcell/gpc/) [38 (link)].

Ishizuka K., Kimura H., Wang C., Xing J., Kushima I., Arioka Y., Oya-Ito T., Uno Y., Okada T., Mori D., Aleksic B, & Ozaki N. (2016). Investigation of Rare Single-Nucleotide PCDH15 Variants in Schizophrenia and Autism Spectrum Disorders. PLoS ONE, 11(4), e0153224.

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Custom TaqMan SNP Genotyping Assay and Statistical Analysis

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We performed a Custom TaqMan® SNP Genotyping assay (Applied Biosystems) for each variant. DNA samples were prepared in 384-well microtiter plates with positive and negative controls. PCR and allelic discrimination analyses were conducted using the Genotyping Master Mix and Sequence Detection System, respectively, according to standard protocols (Applied Biosystems). To determine whether each detected sample carried the variant of interest, positive samples were reconfirmed with Sanger sequencing or with at least one additional experiment. Differences in genotype distribution between cases and controls were calculated with Fisher’s exact test (one-tailed) with a threshold of significance set at p < 0.05.
We computed the effective sample size and statistical power using the web browser program Genetic Power Calculator, which was created by Purcell et al. (http://pngu.mgh.harvard.edu/~purcell/gpc/)^{50 (link)}.

Nawa Y., Kimura H., Mori D., Kato H., Toyama M., Furuta S., Yu Y., Ishizuka K., Kushima I., Aleksic B., Arioka Y., Morikawa M., Okada T., Inada T., Kaibuchi K., Ikeda M., Iwata N., Suzuki M., Okahisa Y., Egawa J., Someya T., Nishimura F., Sasaki T, & Ozaki N. (2020). Rare single-nucleotide DAB1 variants and their contribution to Schizophrenia and autism spectrum disorder susceptibility. Human Genome Variation, 7, 37.

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Replication of Genomic Associations

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Replication cohorts included 2,283 participants from the VIVIT, stenoCardia, and INTERCATH studies. Details about these cohorts are provided in the Supplementary Material.
For replication phase, we selected SNPs with a p-value <5 × 10⁻⁶ from the discovery meta-analysis (rs1485086, rs2376012, rs16835318, and rs17752803) on chromosome 2 and SNP rs1333049 on chromosome 9.
De novo genotyping for replication was performed for the five selected SNPs in VIVIT, stenoCardia and INTERCATH by 5′Nuclease assays using ABI genotyping assays (Applied Biosystems, Darmstadt, Germany). Genotyping was performed in 96-well plates. The Genotyping Master Mix (Applied Biosystems, Darmstadt, Germany) was used in a 7-µl total reaction volume, including 20 ng DNA per reaction. Genotypes were automatically attributed by measuring the allele-specific fluorescence on the ABI 7900HT real-time PCR System (Applied Biosystems, Darmstadt, Germany), using the SDS 2.4 software for allele discrimination (auto caller confidence interval >95%).
Association was tested by a linear regression model assuming additive allele effects in the replication studies. For single SNP replication analysis, we assumed concordant direction of effect and a p-value <0.05. Combined meta-analysis was repeated for the replication cohorts alone.

Zeller T., Seiffert M., Müller C., Scholz M., Schäffer A., Ojeda F., Drexel H., Mündlein A., Kleber M.E., März W., Sinning C., Brunner F.J., Waldeyer C., Keller T., Saely C.H., Sydow K., Thiery J., Teupser D., Blankenberg S, & Schnabel R. (2017). Genome-Wide Association Analysis for Severity of Coronary Artery Disease Using the Gensini Scoring System. Frontiers in Cardiovascular Medicine, 4, 57.

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TaqMan Genotyping Assay Protocol

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Genomic DNA was extracted from peripheral blood using FlexiGene (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Each real-time PCR was performed with a 10 uL reaction volume mix fluid, containing 5 µL Genotyping Master Mix (Applied Biosystems, Waltham, MA, USA), 3.87 uL distilled water, 1 uL DNA fluid (10 ng/uL) and 0.25 uL primer-probe. Amplification reactions were performed using the following program for total 45 cycles: 50 °C for 2 min; 92 °C for 10 min; 95 °C for 15 s; 60 °C for 1 min. We used TaqMan Allelic Discrimination Assays (Applied Biosystems, Foster City, CA, USA) to genotype the SNPs, and the results were read with a 7300 Real-time PCR System (Life Technologies Corp., Carlsbad, CA, USA). The fluorescence level was measured with an Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems, Waltham, USA). The allele frequencies were determined using ABI SDS software. Genotyping was repeated on a random 10% sample to confirm the results of the original run by the laboratory technicians, who were blinded to the original results. The estimated genotyping error rate was less than 1%.

Ho K.J., Chen T.H., Yang C.C., Chuang Y.C, & Chuang H.Y. (2021). Interaction of Smoking and Lead Exposure among Carriers of Genetic Variants Associated with a Higher Level of Oxidative Stress Indicators. International Journal of Environmental Research and Public Health, 18(16), 8325.

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SNP Genotyping for Genetic Profiling

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Genomic DNA has been extracted previously from blood samples with the QIAamp DNA Blood Midi kit (Qiagen, Venlo, The Netherlands), and from 122 fresh-frozen primary tumour samples with the High Pure PCR Template Preparation Kit (Roche, Vienna, Austria) as described [27] (link), [28] (link). Genotyping of SNP rs1800449 (G473A; c.473G>A; Arg158Gln; R158Q) in the LOX gene was performed by TaqMan PCR with Genotyping Mastermix and allele-specific, fluorescently labeled probes on a 7500 fast instrument following the manufacturer’s instructions (Applied Biosystems, Brunn/Gebirge, Austria; Assay-ID #C___7574719_10). 20 ng of genomic DNA were used per reaction in a reaction volume of 10 µl.

Friesenhengst A., Pribitzer-Winner T, & Schreiber M. (2014). Association of the G473A Polymorphism and Expression of Lysyl Oxidase with Breast Cancer Risk and Survival in European Women: A Hospital-Based Case-Control Study. PLoS ONE, 9(8), e105579.

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Genomic DNA Extraction and Genotyping

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Briefly, peripheral blood samples were collected from subjects in 6-ml EDTA vacuum tubes. Genomic DNA was extracted from peripheral blood using the standard phenol–chloroform extraction method (29 (link)). The final concentrations of all DNA samples were 20 ng/μl with good purity (OD260/OD280 = 1.8–2.0) and stored at −80°C until genotyping. The TaqMan real-time quantitative PCR (q-PCR) method was used to test DNA samples using the TaqMan® Genotyping Assay (cat#25800731, cat#27481263, cat#86363451, cat#29122242, Applied Biosystems, Shanghai, China) on an ABI ViiA™ 7 sequence detection system instrument (Applied Biosystems). PCR was performed in 25-μL reactions containing Genotyping Master Mix (Applied Biosystems, Shanghai, China), TaqMan® Genotyping Assay, and DNA template. Each 96-well-plate was quality-controlled with two no-template controls. The PCR program for the amplification was as follows: 95°C for 10 min and then 40 cycles of denaturation (95°C for 15 s) and annealing (60°C for 1 min). We further randomly selected 10% samples for each of the selected SNPs for re-genotyping, and the results were 100% concordant.

Chen J., Zhang M., Zhou C., Ding Y., Fan N, & He H. (2020). Association Analysis of Neuronal Nitric Oxide Synthase 1 Gene Polymorphism With Psychopathological Symptoms in Chronic Ketamine Users. Frontiers in Psychiatry, 11, 580771.

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Genotyping of Putative Deleterious Variant

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The effective sample size and statistical power was computed using the web browser
program, Genetic Power Calculator, developed by Purcell et al. (http://pngu.mgh.harvard.edu/~purcell/gpc/).^{52 (link)} An ABI PRISM 7900HT Sequence Detection System (Applied
Biosystems) and TaqMan assays with custom probes were used to genotype a putative
deleterious variant. Custom probe sequences are indicated in Supplementary Table S4. Each 384-microtiter plate contained two
non-template controls and two samples with the variant. The reactions and data
analysis were performed using Genotyping Master Mix and Sequence Detection
Systems, respectively, according to standard protocols (Applied Biosystems).
Differences in genotype distribution between cases and controls were tested with
one-sided, Fisher’s exact tests.

Ishizuka K., Fujita Y., Kawabata T., Kimura H., Iwayama Y., Inada T., Okahisa Y., Egawa J., Usami M., Kushima I., Uno Y., Okada T., Ikeda M., Aleksic B., Mori D., Someya T., Yoshikawa T., Iwata N., Nakamura H., Yamashita T, & Ozaki N. (2017). Rare genetic variants in CX3CR1 and their contribution to the increased risk of schizophrenia and autism spectrum disorders. Translational Psychiatry, 7(8), e1184-.

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Genotyping CYP3A5 Polymorphisms

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Secondary use samples of extracted DNA were obtained from the University of Michigan histocompatibility laboratory for patients who met the inclusion criteria. Commercial Taqman assays were used as described previously, with minor modifications.15 (link) Briefly, reactions were carried out using 10 ng of DNA with Genotyping Master Mix (Applied Biosystems) in an iCycler real-time thermocycler (BioRad) for 40 cycles. Genotyping was performed for the following single nucleotide polymorphisms (SNP) CYP3A5*3 (rs776746), CYP3A5*6 (rs10264272), and CYP3A5*7 (rs41303343) (Thermo Fisher Scientific, Waltham, MA). Call rates for all SNPs were >99%; 10% of samples were retested and results were 100% concordant. CYP3A5 genotypes were grouped into the corresponding clinical phenotype for analysis: CYP3A5 expressers (those with 1 or zero variant alleles) and CYP3A5 non-expressers (those with 2 variant alleles).

Pasternak A.L., Marshall V.D., Gersch C.L., Rae J.M., Englesbe M, & Park J.M. (2021). Evaluating the Impact of CYP3A5 Genotype on Post-Transplant Healthcare Resource Utilization in Pediatric Renal and Heart Transplant Recipients Receiving Tacrolimus. Pharmacogenomics and Personalized Medicine, 14, 319-326.

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Genomic DNA Extraction and Genotyping

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Genomic DNA was extracted from peripheral blood samples (3 mL) by using the Blood Genomic Prep Mini Spin Kit (GE Heathcare, Buckinghamshire, UK). Genotyping analyses were conducted by using TaqMan real-time polymerase chain reaction (RT-PCR) assays in an ABI PRISM 7500 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). Each reaction contained 1 µL of genomic DNA (20 ng/µL); 0.5 µL of probe, either C_7586662_10 for rs1128503 or C__7586657_20 for rs1045642 (Applied Biosystems); 5µL of Genotyping Master Mix (Applied Biosystems); and 3.5 µL of water. All the experiments were carried out in 96-well plates, including a nontemplate control and at least two positive controls.

Delou J.M., Vignal G.M., Índio-do-Brasil V., Accioly M.T., da Silva T.S., Piranda D.N., Sobral-Leite M., de Carvalho M.A., Capella M.A, & Vianna-Jorge R. (2017). Loss of constitutive ABCB1 expression in breast cancer associated with worse prognosis. Breast Cancer : Targets and Therapy, 9, 415-428.

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Plasma TNF-α Genotyping and Quantification

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From all study participants, 5 ml of whole blood was obtained (stored in − 80 °C). DNA Blood Mini Kit (Qiagen, Canada) was used to DNA isolation. DNA quality and quantity were evaluated with the use of NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, USA). Genotyping was performed using a real-time PCR method with allele-discriminating software. The Genotyping Master Mix and TaqMan probes (Applied Biosystems, USA) specific for studied TNF-α SNP (Thermo Fisher Scientific, USA, SNP ID: C_7514871_10, cat. no. 4351379) were used to DNA amplification under the condition of manufacturer protocol provided with kit in RT7500 Real-time PCR device (Applied Biosystems, USA). Because we were interested in the assessment of the generalised inflammation process (TNF-α in circulation) instead of local changes (TNF-α in saliva), we decided to use plasma instead of saliva as a test material. The plasma TNF-α level was assessed using TNF alpha Human ELISA Kit Ultrasensitive (Thermo Fisher Scientific, USA, cat. no. KHC3014). The detection range was 0.2–32 pg/mL, and the sensitivity was equal to the minimal detectable dose of this kit (< 0.09 pg/mL).

Mlak R., Powrózek T., Brzozowska A., Homa-Mlak I., Mazurek M., Gołębiowski P., Sobieszek G, & Małecka-Massalska T. (2019). The relationship between TNF-α gene promoter polymorphism (− 1211 T > C), the plasma concentration of TNF-α, and risk of oral mucositis and shortening of overall survival in patients subjected to intensity-modulated radiation therapy due to head and neck cancer. Supportive Care in Cancer, 28(2), 531-540.

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