Custom taqman snp genotyping assay

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, United Kingdom

Custom TaqMan SNP Genotyping Assays are a tool for analyzing single nucleotide polymorphisms (SNPs) in genetic samples. These assays provide a standardized and streamlined method for SNP detection and genotyping.

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TaqMan assays (1810 citations) TaqMan genotyping assays (278 citations) TaqMan SNP assay (59 citations) TaqMan SNP genotyping (24 citations) SNP genotyping assay (20 citations) Custom Taqman assays (17 citations) Genotyping assays (3 citations)

88 protocols using custom taqman snp genotyping assay

Genotyping kdr Mutations in Mosquitoes

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We performed independent genotyping reactions for each kdr site based on a qPCR approach using the Custom TaqMan SNP Genotyping Assay (ThermoFisher) (Macoris et al., 2018 (link)) (see Supplementary Table S1 for the primer and probe sequences for each assay). Reactions consisted of 1X TaqMan Genotyping Master Mix (ThermoFisher), 1X of the respective Custom TaqMan SNP Genotyping Assay, 20 ng of DNA and ultra-pure water q. s. 10 µl, run in a QuantStudio 6 Flex (Applied Biosystems), under standard conditions: 45 cycles with a DNA denaturation step (95°C for 15 s) and primer and probe annealing, followed by DNA polymerization (60°C for 1 min). The genotypes were obtained by the online software Genotype Analysis Module V3.9 (Applied Biosystems, Thermo Fischer cloud platform). We evaluated the kdr sites 1016 (V1016I) and 1534 (F1534C) in both populations and genotypes were determined as detailed elsewhere (Macoris et al., 2018 (link)) (Supplementary Table S2).

Cosme L.V., Lima J.B., Powell J.R, & Martins A.J. (2022). Genome-wide Association Study Reveals New Loci Associated With Pyrethroid Resistance in Aedes aegypti. Frontiers in Genetics, 13, 867231.

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Genotyping of rs12979860 and rs368234815

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Genomic DNA was extracted from whole blood using the Qiagen Flexigene DNA Kit (Qiagen). The rs12979860 SNP was genotyped by allelic discrimination using a TaqMan Custom SNP Genotyping Assay (Applied Biosystems) on an ABI 7500 Real-Time PCR System (Applied Biosystems). rs368234815 was genotyped by Sanger sequencing using BigDye Terminator v3.1 (Applied Biosystems) according to the manufacturer’s protocol, or by allelic discrimination using a TaqMan Custom SNP Genotyping Assay (Applied Biosystems) as previously described.^{15 (link)}

Noureddin M., Rotman Y., Zhang F., Park H., Rehermann B., Thomas E, & Liang T. (2015). Hepatic expression levels of interferons and interferon-stimulated genes in patients with chronic hepatitis C: A phenotype–genotype correlation study. Genes and immunity, 16(5), 321-329.

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SNP Genotyping for APOE Alleles

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The Custom Taqman SNP Genotyping Assays (Applied Biosystems, Foster City, USA) were used to prescreen for the rs405509 genotype in the participants. The extra two SNPs, rs429358 and rs7412, which jointly defined the APOE ε2 (with haplotype of rs429358-rs7414: T-T), ε3 (T-C), and ε4 alleles (C-C), were also genotyped. The sample success rates for all of the SNPs were 100%, and the reproducibility of all of the genotyping was 100% according to a duplication analysis of at least 10% of the genotypes. According to the Hapmap database, the rs405509 is in high LD (D′>0.8) with rs7412 but not with rs429358. For both rs405509 and APOE, we did not encounter significant deviations from Hardy-Weinberg equilibrium at an alpha level of 0.05. Rs405509-APOE combined genotypes were defined by Plink (31 (link)). Totally, there were 12 combined genotypes (Table S1).

Ma C., Zhang Y., Li X., Zhang J., Chen K., Liang Y., Chen Y., Liu Z, & Zhang Z. (2016). Is there a significant interaction between APOE rs405509 T/T, and e4 genotypes on cognitive impairment and gray matter volume?. European journal of neurology, 23(9), 1415-1425.

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Thr161Lys Mutation Analysis in hiPSC-Derived CMs

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RNA samples were collected and extracted from hiPSC-derived CMs (09703.HCMJp) with Norgen’s Total RNA Purification Plus Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturer’s instructions. A total of 50–100 ng of RNA was transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies Ltd., Waltham, MA, USA). The expression of the Thr161Lys mutation at the mRNA level in the hiPSC-derived CMs was studied using custom TaqMan SNP genotyping assays (Applied Biosystems, Life Technologies Ltd., Waltham, MA, USA) as above.

Valtonen J., Prajapati C., Cherian R.M., Vanninen S., Ojala M., Leivo K., Heliö T., Koskenvuo J, & Aalto-Setälä K. (2023). The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity. Biomedicines, 11(6), 1558.

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ESR1 Mutation Detection Protocols

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We used LBx^® Probe ESR1 E380Q (#65116) as the detection probe for ESR1 E380Q (Riken Genesis, Tokyo, Japan) and Custom TaqMan SNP Genotyping assays (Applied Biosystems, Foster City, CA, USA) for the detection of other ESR1 LBD mutations (Y537S, Y537N, Y537C, and D538G), as described previously [20 ].

Takeshita T., Yamamoto Y., Yamamoto-Ibusuki M., Sueta A., Tomiguchi M., Murakami K., Omoto Y, & Iwase H. (2017). Prevalence of ESR1 E380Q mutation in tumor tissue and plasma from Japanese breast cancer patients. BMC Cancer, 17, 786.

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DNA Extraction and Genotyping of p.R4810K

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Peripheral blood (2–10 ml) was collected from all participants. DNA was extracted from the blood samples using a QIAamp DNA Blood Mini Kit (Qiagen, Germantown, Maryland, USA) according to the manufacturer’s instructions. The quality and concentration of the extracted DNA were measured using an Infinite M200 PRO (TECAN, Kanagawa, Japan). The DNA was stored in a freezer at -30 °C until analysis. Genotyping of the p.R4810K mutation was conducted for all the participants using a TaqMan probe (Custom TaqMan SNP Genotyping Assays; Applied Biosystems, Foster City, CA, USA) and a 7300/7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions.

Mineharu Y., Nakamura Y., Sato N., Kamata T., Oichi Y., Fujitani T., Funaki T., Okuno Y., Miyamoto S., Koizumi A, & Harada K.H. (2022). Increased abundance of Ruminococcus gnavus in gut microbiota is associated with moyamoya disease and non-moyamoya intracranial large artery disease. Scientific Reports, 12, 20244.

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Genetic Profiling of ELN Variants

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Genomic DNA was extracted from peripheral blood using standard methodology. Genotyping was performed using the TaqMan single nucleotide polymorphism (SNP) Genotyping Assays or Custom TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA) on StepOnePlus Real-Time PCR System (Applied Biosystems) in accordance with the supplier’s recommendations. Examined SNPs were ELN rs868005, rs884843, rs2301995, rs13239907, rs2856728, which were examined in our previous study [8 (link)]. Briefly, SNPs were selected from the HapMap Project [20 (link)] database for the JPT (Japanese in Tokyo) population using the Tagger tool [21 (link)], with minor allelic frequencies (MAF) above 10% that correlated at D’ > 0.8 with all ELN SNPs of > 10% MAF.

Yanagisawa S., Sakurada Y., Miki A., Matsumiya W., Imoto I, & Honda S. (2015). The Association of Elastin Gene Variants with Two Angiographic Subtypes of Polypoidal Choroidal Vasculopathy. PLoS ONE, 10(3), e0120643.

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Genotyping of Human DNA Samples

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Trained research assistants obtained DNA samples from participants by collecting buccal cells using the Epicentre Catch-All Collection Swabs or by collecting saliva using the Oragene DNA Self-Collection kits. DNA was extracted and prepared for polymerase chain reaction (PCR) amplification using the Epicentre BuccalAmp DNA Extraction Kit (Epicentre, Cat. No. BQ090155C). Genotyping was then performed using established protocols. SNP genotyping was conducted using Applied Biosystems Custom Taqman SNP Genotyping Assays. The products of these analyses were then analyzed using endpoint allelic discrimination. Genotypes were identified and sequenced with the Beckman-Coulter CEQ8000 semiautomated florescent sequencing system, which utilizes Fragment Analysis Application and associated software. All samples were genotyped twice for quality control. Human DNA from cell lines were purchased from Coriell Cell Repositories for each genotype and used as control samples using DTCS chemistry on an ABI 3130×l. These cell lines and a no template control were run with study samples representing 9% of the total data output. Samples that were not able to be genotyped to a 95% or greater confidence level were repeated under the same procedures up to four times.

Cicchetti D., Rogosch F.A., Hecht K.F., Crick N.R, & Hetzel S. (2014). Moderation of maltreatment effects on childhood borderline personality symptoms by gender and oxytocin receptor and FK506 binding protein 5 genes. Development and psychopathology, 26(3), 831-849.

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Genetic Profiles in Liver Transplant

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Genomic DNA was extracted from the peripheral blood mononuclear cells of donors and recipients before LDLT by use of a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was performed to detect the single nucleotide polymorphisms (SNP) VDR rs2228530 and CYP2R1 rs10741657 with a ready-to-use, manufacturer-validated, predesigned allele-discriminating TaqMan SNP assay for polymerase chain reaction (PCR) amplification in clear optical 96-well plates on a 7500 Fast Real-Time PCR system (Applied Biosystems International, Foster City, CA, USA), according to the manufacturer’s instructions. The VDR SNP rs2228570 and CYP2R1 rs10741657 genotypes were also studied in liver tissues via selective graft biopsies after LDLT on the same 7500 Fast Real-Time PCR system with Custom TaqMan SNP genotyping assays (Applied Biosystems) for allele discrimination. Both the serum and liver graft biopsy tissue SNPs were selected according to VDR rs2228530 AA/AG/GG and CYP2R1 rs10741657 AA/AG/GG allele frequencies and functional clinical implications. All genotypes were assayed in duplicate to assess intraassay precision. Genetic modification is defined as the difference of the SNP of VDR rs2228570 and CYP2R1 rs10741657 genotypes before and after LDLT.

Lin S.H., Wang C.C., Huang K.T., Chen K.D., Hsu L.W., Eng H.L, & Chiu K.W. (2022). Liver Graft Pathology and Low Serum 25-Hydroxyvitamin D after Living Donor Liver Transplantation. Metabolites, 12(5), 388.

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Genetic Polymorphism Analysis Protocol

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DNA was isolated from peripheral blood by a standard desalting method. The IL28B rs12979860 SNP was determined using Custom Taqman SNP Genotyping Assays (Applied Biosystems, Life Technologies, Foster, CA, USA). The IL10R −1087 (also known as IL10R −1082) (rs1800896) promoter region was required for the formation of the EcoNI recognition sequence 5′-AAGACAACACTACTAAGGCT-3′; the lower primer was 5′-TAAATATCCTCAAAGTTCC-3′. After cutting the amplified product (584 bp) by EcoNI, homozygote GG was identified by two fragments 315 and 279 bp, while heterozygote AG had 310, 280, 252 and 28 bp fragments and homozygote AA had 310, 252 and 28 bp fragments [27 (link)].

Pár A., Pár G., Tornai I., Szalay F., Várszegi D., Fráter E., Papp M., Lengyel G., Fehér J., Varga M., Gervain J., Schuller J., Nemes Z., Péterfi Z., Tusnádi A., Hunyady B., Haragh A., Szinku Z., Vincze Á., Szereday L., Kisfali P, & Melegh B. (2014). IL28B and IL10R −1087 polymorphisms are protective for chronic genotype 1 HCV infection and predictors of response to interferon-based therapy in an East-Central European cohort. BMC Research Notes, 7, 12.

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