Type it hrm reagents

Manufactured by Qiagen

Sourced in United States

The Type-It HRM reagents are a set of molecular biology reagents designed for high-resolution melt (HRM) analysis. The reagents are intended to be used for the detection and analysis of genetic variations, such as single nucleotide polymorphisms (SNPs), in DNA samples. The core function of the Type-It HRM reagents is to provide the necessary components for performing HRM analysis, which is a technique used to identify DNA sequence differences based on the melting behavior of the DNA molecules.

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6 protocols using type it hrm reagents

Genotyping Protocols for Genetic Comparison

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Case-control comparisons for Oulu, Kuopio and Tampere cohorts were performed by High Resolution Melt (HRM) analysis (CFX96, Bio-Rad, Hercules, CA, USA) using Type-It HRM reagents (Qiagen, Hilden, Germany). A positive control was included in all analyses, and samples with differing melting curves were further confirmed by Sanger sequencing (ABI3130xl, Applied Biosystem, Foster City, CA, USA). The Helsinki cohorts were genotyped using Custom TaqMan SNP Genotyping Assays and TaqMan Genotyping MasterMix (ThermoFisher Scientific, Waltham, MA, USA). PCR was performed on 7500 Fast Real-Time PCR System or 9800 Fast Thermal Cycler and genotypes were analyzed on 7500 Fast System SDS software v1.3.1 or 7500 software v2.0.6 (Applied Biosystems, Waltham, MA, USA). Heterozygous carriers were included as positive controls in all analyses.

Mantere T., Tervasmäki A., Nurmi A., Rapakko K., Kauppila S., Tang J., Schleutker J., Kallioniemi A., Hartikainen J.M., Mannermaa A., Nieminen P., Hanhisalo R., Lehto S., Suvanto M., Grip M., Jukkola-Vuorinen A., Tengström M., Auvinen P., Kvist A., Borg Å., Blomqvist C., Aittomäki K., Greenberg R.A., Winqvist R., Nevanlinna H, & Pylkäs K. (2017). Case-control analysis of truncating mutations in DNA damage response genes connects TEX15 and FANCD2 with hereditary breast cancer susceptibility. Scientific Reports, 7, 681.

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Genotyping and Sequencing of CHEK2 Variants

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Genotyping was performed for DNA samples extracted from peripheral blood by using high-resolution melt analysis (CFX96, Bio-Rad, Hercules, CA, USA) with Type-It HRM reagents (Qiagen, Hilden, Germany). Verification of all detected CHEK2 p.(Asp438Tyr) variants were confirmed with Sanger sequencing (ABI3130xl, Applied Biosystem, Foster City, CA, USA). All identified p.(Asp438Tyr) carriers were genotyped for CHEK2 c.1100delC (MAF 0.01 in North Ostrobothnia, SISu) with direct sequencing (ABI). The used primers are shown in Online Resource Supplementary Table 1.

Kumpula T.A., Koivuluoma S., Soikkonen L., Vorimo S., Moilanen J., Winqvist R., Mantere T., Kuismin O, & Pylkäs K. (2023). Evaluating the role of CHEK2 p.(Asp438Tyr) allele in inherited breast cancer predisposition. Familial Cancer, 22(3), 291-294.

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ATM Variant Genotyping and Validation

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Genotyping was performed for DNA samples extracted from peripheral blood by using high‐resolution melt analysis (CFX96, Bio‐Rad, Hercules, CA, USA) with Type‐It HRM reagents (Qiagen, Hilden, Germany). Verification of all detected ATM c.7570G>C variants were confirmed with Sanger sequencing in Biocenter Oulu Sequencing Center (ABI3130xl, Applied Biosystem, Foster City, CA, USA).

Kankuri‐Tammilehto M., Tervasmäki A., Kraatari‐Tiri M., Rahikkala E., Pylkäs K, & Kuismin O. (2022). ATM c.7570G>C is a high‐risk allele for breast cancer. International Journal of Cancer, 152(3), 429-435.

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Genotyping of FANCM Mutations

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Genotyping of the FANCM c.5791C>T mutation for the Helsinki and Tampere sample sets was performed with Sequenom MassARRAY system using iPLEX Gold assays (Sequenom) at FIMM (University of Helsinki). Variants c.4025_4026delCT and c.5293dupA were genotyped with TaqMan real-time PCR. 7500 Fast Real Time system was utilized by using TaqMan SNP Genotyping Custom assays and TaqMan Genotyping MasterMix (Applied Biosystems). Genotype calling was performed with 7500 RealTime PCR System and 7500 software (version 2.06, Applied Biosystems). Genotyping for the FANCM c.5101C>T mutation was performed as previously described [12 (link)]. Positive controls were used in all analyses and all mutations were confirmed with Sanger sequencing. It is to be noted that one patient from the Helsinki dataset (counted as one in the analysis) carries both c.5101C>T and c.5791C>T mutations. Unfortunately, we were not able to determine whether the mutations are in cis or in trans; however, if they were in cis, this genotype would be extremely rare.
FANCM c.5791C>T and c.5101C>T screening for Oulu and Kuopio sample sets was performed using High Resolution Melt analysis (CFX96, Bio-Rad) with Type-it HRM reagents (Qiagen). Positive control DNA was included in all analyses, and samples with positive-like or differing melting curves were validated by Sanger sequencing (ABI3130xl, Applied Biosystems).

Kiiski J.I., Tervasmäki A., Pelttari L.M., Khan S., Mantere T., Pylkäs K., Mannermaa A., Tengström M., Kvist A., Borg Å., Kosma V.M., Kallioniemi A., Schleutker J., Bützow R., Blomqvist C., Aittomäki K., Winqvist R, & Nevanlinna H. (2017). FANCM mutation c.5791C>T is a risk factor for triple-negative breast cancer in the Finnish population. Breast Cancer Research and Treatment, 166(1), 217-226.

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Variant Genotyping and Confirmation

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Variants passing the filtering criteria were genotyped using Agena Bioscience MassARRAY System (Sequenom Inc., FIMM) and High-Resolution Melt (HRM) analysis (CFX96, Bio-Rad) with Type-It HRM reagents (Qiagen). Sanger sequencing (ABI3500xL Genetic Analyzer, Applied Biosystems) was used for confirmation of the variants.

Koivuluoma S., Tervasmäki A., Kauppila S., Winqvist R., Kumpula T., Kuismin O., Moilanen J, & Pylkäs K. (2021). Exome sequencing identifies a recurrent variant in SERPINA3 associating with hereditary susceptibility to breast cancer. European journal of cancer (Oxford, England : 1990), 143.

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NTHL1 p.Q90* Variant Genotyping

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Genotyping was performed for DNA samples extracted from peripheral blood by using high‐resolution melt analysis (CFX96, Bio‐Rad) with Type‐It HRM reagents (Qiagen). All assays included heterozygous and homozygous NTHL1 p.Q90* (NM_002528.5) genomic DNA samples as positive controls. Verification of all detected p.Q90* variants were confirmed with Sanger sequencing (ABI3130xl, Applied Biosystems).

Kumpula T., Tervasmäki A., Mantere T., Koivuluoma S., Huilaja L., Tasanen K., Winqvist R., de Voer R.M, & Pylkäs K. (2020). Evaluating the role of NTHL1 p.Q90* allele in inherited breast cancer predisposition. Molecular Genetics & Genomic Medicine, 8(11), e1493.

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