Genemapper 5

Manufactured by Thermo Fisher Scientific

Sourced in United States

GeneMapper 5.0 is a software application developed by Thermo Fisher Scientific for DNA fragment analysis. It is designed to analyze DNA fragments generated from various genetic analysis techniques, including capillary electrophoresis-based sequencing and fragment analysis.

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GeneMapper (100 citations) GeneMapper Software 5 (49 citations) GeneMapper version 5.0 software (14 citations) GeneMapper software version 5.0 (7 citations) GeneMapper 3.5 program (2 citations) GeneMapper™ v1.5) (2 citations)

67 protocols using genemapper 5

Detecting Splice Variants in Transfected HEK 293T Cells

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RT-PCR from cDNA obtained from HEK 293 T cells (ATCC, Teddington, UK) transfected with plasmid constructs harboring the mutant c.1663-1205A-allele was performed with a 5′ FAM (6-carboxyfluorescein) labeled forward primer located in exon 14 (5-CCGTTCTCTATTTGCCTGGT-3´) and a reverse primer located in the pSPL3 tat2 exon (5-GATCCATTCGACCAATTCACT-3´) using the AmpliTaqGold polymerase (Thermo Fisher Scientific, Karlsruhe, Germany). FAM-labeled RT-PCR products were diluted 1:100 in water, mixed with 1 μl of GeneScan ROX500 size standard (Life Technologies, Darmstadt, Germany) and 8 μl of Hi-Di Formamide (Life Technologies) in a total volume of 10 μl. Mixes were separated by capillary electrophoresis on an ABI 3130XL Genetic Analyzer instrument (Life Technologies). The area-under-the-curve (AUC) was calculated with GeneMapper 5 (Life Technologies) software. Ratios of splicing products were determined as the AUC for individual peaks divided by the sum of AUC of all differentially spliced products.

Weisschuh N., Sturm M., Baumann B., Audo I., Ayuso C., Bocquet B., Branham K., Brooks B.P., Catalá-Mora J., Giorda R., Heckenlively J.R., Hufnagel R.B., Jacobson S.G., Kellner U., Kitsiou-Tzeli S., Matet A., Sampol L.M., Meunier I., Rudolph G., Sharon D., Stingl K., Streubel B., Varsányi B., Wissinger B, & Kohl S. (2019). Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation. Human mutation, 41(1), 255-264.

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Plasmid Linearization and Sequencing

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Plasmid pEGFP-BSf (500 ng) pre-hybridized with bisLNA (4 μM, 72 h) was digested with S1 nuclease following protocol described above. After digestion, the samples were purified using Spin columns (Qiagen PCR purification kit) according to the manufacturers protocol. The plasmids were subsequently linearized using NdeI and SacI (Fermentas). Primer extension was carried out under the following conditions: [2 mM MgCl₂, 3 U Taq polymerase (Fermentas), 2.5 nM primer, 2 mM of each dNTP. Ten minutes at 94°C, 29 cycles of 1 min at 94°C, 2 min at 45°C, 3 min at 72°C and finally 10 min at 72°C. Primer sequence: 5′ 6FAM-AAA TGG GCG GTA GGC GT 3′ (Cybergene)]. Plasmid was sequenced using Thermo Sequenase Primer Dye Manual Cycle Sequencing kit (Affymetrix). For each sample, 5 μl of the PCR reaction, 4.75 μl of HiDi (Life Technologies) and 0.25 μl of GeneScan 600-LIZ (Life Technologies) were analyzed using ABI 3730 DNA Analyzer (injection voltage 3 kV, 30 s). The electropherograms were analyzed with GeneMapper 5 (Life Technologies) and horizontally aligned in the sample plot window through the use of the size standard. The reactions from the Thermo Sequenase kit were superimposed and aligned to the digestion samples to read the sequence.

Geny S., Moreno P.M., Krzywkowski T., Gissberg O., Andersen N.K., Isse A.J., El-Madani A.M., Lou C., Pabon Y.V., Anderson B.A., Zaghloul E.M., Zain R., Hrdlicka P.J., Jørgensen P.T., Nilsson M., Lundin K.E., Pedersen E.B., Wengel J, & Smith C.I. (2016). Next-generation bis-locked nucleic acids with stacking linker and 2′-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes. Nucleic Acids Research, 44(5), 2007-2019.

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Microsatellite Genotyping of Brook Trout

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Genomic DNA was extracted using the Mag‐Bind ® Tissue DNA Kit (Omega Bio‐tek) with the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific) as well as the Puregene (Gentra Systems, Inc.) methods, following the manufacturers’ protocols. Samples were genotyped at 12 microsatellite markers developed in Brook Trout: SfoB52, SfoC38, SfoC113, SfoD75, SfoD100, SfoC28, SfoC86, SfoC88, SfoC129, SfoC24, SfoC115, and SfoD91 (King, Lubinski, Burnham‐Curtis, Stott, & Morgan, 2012). Markers were combined into three multiplex reactions for PCR amplification and fragment analysis. Each 15 µl PCR consisted of 1.5 µl genomic DNA extract, 1.5× PCR buffer, 3.75 mM MgCl₂, 0.3175 mM dNTPs, 0.08–0.18 µM of each primer, and 0.08 units/µl GoTaq® Flexi DNA polymerase (Promega). The amplification protocol followed that of King et al. (2012). PCR products were then visualized on an ABI 3130XL genetic analyzer (Life Technologies), and alleles were scored with GeneMapper 5 (Life Technologies) by two independent readers. As a quality control measure, 10% of the samples were re‐extracted and genotyped to compare against the original data.

Beer S.D., Cornett S., Austerman P., Trometer B., Hoffman T, & Bartron M.L. (2019). Genetic diversity, admixture, and hatchery influence in Brook Trout (Salvelinus fontinalis) throughout western New York State. Ecology and Evolution, 9(13), 7455-7479.

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Quantitative RT-PCR Analysis of Genetic Variants

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Four hundred ng of total RNA isolated from blood samples was used for cDNA synthesis using random hexamers and the Maxima H Minus Reverse Transcriptase according to the manufacturer’s protocol (Thermo Fisher Scientific, Carlsbad, USA). For the analysis of variants c.1065+6T>C and c.1212+4del, reverse transcription polymerase chain reaction (RT-PCR) was performed using 2 μl cDNA, a forward primer located in exon 7 (5´- TGGAACGATTAGAAAAGGAGAACAAAG-3´), a 5′ FAM (6-carboxyfluorescein) labeled reverse primer located in exon 14 (5´- CCATGAGGGTCCATTTGACT-3´), and standard PCR conditions (35 cycles). For the analysis of variant c.1516+3A>G, a forward primer located in exon 11 (5´- GAACTTCGAATGAGGAAAAATGTGA-3´) and a 5′ FAM (6-carboxyfluorescein) labeled reverse primer located in exon 17 (5´- TGTTGTTCAACAGACTCTCGTACCAT-3´) was used.
FAM-labeled RT-PCR products were mixed with 0.5 μl of GeneScan ROX500 size standard (Life Technologies, Darmstadt, Germany) and 8.5 μl of Hi-Di Formamide (Life Technologies) in a total volume of 10 μl. Mixes were separated by capillary electrophoresis on an ABI 3130XL Genetic Analyzer instrument (Life Technologies). The area-under-the-curve (AUC) was calculated with GeneMapper 5 (Life Technologies) software. Ratios of RT-PCR products were determined as the AUC for individual peaks divided by the sum of AUC of all peaks.

Weisschuh N., Schimpf-Linzenbold S., Mazzola P., Kieninger S., Xiao T., Kellner U., Neuhann T., Kelbsch C., Tonagel F., Wilhelm H., Kohl S, & Wissinger B. (2021). Mutation spectrum of the OPA1 gene in a large cohort of patients with suspected dominant optic atrophy: Identification and classification of 48 novel variants. PLoS ONE, 16(7), e0253987.

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RT-PCR Splicing Product Analysis

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First strand cDNA synthesis was performed using 2 μg of total RNA and random hexamer primers. Subsequent PCR was performed with a 5′ FAM (6-carboxyfluorescein) labeled forward primer and using the QIAGEN Multiplex PCR Kit (Qiagen, Hilden, Germany). FAM-labeled RT-PCR products were diluted 1:10 in water; mixed with 1 μl of GeneScan ROX500 size standard (Life Technologies, Darmstadt, Germany) and 8 μl of Hi-Di Formamide (Life Technologies) in a total volume of 10 μl. Mixes were separated by capillary electrophoresis on an ABI 3130XL Genetic Analyzer instrument (Life Technologies). The area-under-the-curve (AUC) was calculated with GeneMapper 5 (Life Technologies) software. Ratios of splicing products were determined as the AUC for individual peaks divided by the sum of AUC of all differentially spliced products.

Buena-Atienza E., Rüther K., Baumann B., Bergholz R., Birch D., De Baere E., Dollfus H., Greally M.T., Gustavsson P., Hamel C.P., Heckenlively J.R., Leroy B.P., Plomp A.S., Pott J.W., Rose K., Rosenberg T., Stark Z., Verheij J.B., Weleber R., Zobor D., Weisschuh N., Kohl S, & Wissinger B. (2016). De novo intrachromosomal gene conversion from OPN1MW to OPN1LW in the male germline results in Blue Cone Monochromacy. Scientific Reports, 6, 28253.

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STR Profiling of iPSCs and PBMCs

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Genomic DNA of iPSCs (p52) and patient’s PBMCs was extracted using Quick-DNA Miniprep Plus Kit (Zymo Research). STRs were amplified using the PowerPlex16 (Promega) system and resolved on a ABI 3730xl Genetic Analyzer (Thermo Fischer Scientific). GeneMapper 5.0 (Thermo Fischer) software was used to call the genotype at each locus and compare between peripheral blood and iPSC samples.

Contreras J., Alonzo M., Ye S., Lin H., Hernandez-Rosario L., McBride K.L., Texter K., Garg V, & Zhao M.T. (2022). Generation of an induced pluripotent stem cell line NCHi003-A from a 11-year-old male with pulmonary atresia with intact ventricular septum (PA-IVS). Stem cell research, 64, 102893.

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iPSC and PBMC Genomic DNA Profiling

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Genomic DNA was extracted from iPSCs (passages 14–15) and PBMCs using the Quick-DNA Miniprep Plus Kit (Zymo Research). The PowerPlex 16 System (Promega) was then utilized to amplify genomic materials according to the manufacturer’s instructions. Samples were sent for capillary sequencing using an ABI 3730xl Genetic Analyzer (Thermo Fisher Scientific). GeneMapper 5.0 (Thermo Fisher Scientific) software was used to analyze the sequencing data for allele callings for 16 loci per sample. Only strong allele calling signals were considered for analysis.

Alonzo M., Contreras J., Ye S., Lin H., Hernandez-Rosario L., McBride K.L., Texter K., Garg V, & Zhao M.T. (2022). Characterization of an iPSC line NCHi006-A from a patient with hypoplastic left heart syndrome (HLHS). Stem cell research, 64, 102892.

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STR Profiling of iPSCs and PBMCs

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iPSC and PBMC Genomic DNA Profiling

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Microsatellite-based Fluorescent PCR Assay

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A fluorescent PCR assay was used to amplify the EmsB microsatellite, following a previous protocol [34 (link)]. Fragment analysis of PCR products was performed by capillary electrophoresis on an automatic sequencer (ABI Prism 310 and ABI 3500/3730; Life Technologies, CA). The resulting EmsB electropherograms were composed of several peaks between 209 and 241 bp. The size (base-pair length) and height (fluorescent signal intensity) of peaks present in each EmsB electropherogram were determined using GeneMapper 5.0 (Life Technologies, CA). Characterization of each EmsB profile was performed as described in the EmsB guidelines from the EWET database (https://ewet-db.univ-fcomte.fr/) [40 (link)]. Briefly, peaks below 10% of the sample's maximum peak height were considered artefacts and discarded. To normalize raw data, the height of each peak was divided by the sum of the height of all peaks of a given sample.

Santa M.A., Umhang G., Klein C., Grant D.M., Ruckstuhl K.E., Musiani M., Gilleard J.S, & Massolo A. (2023). It's a small world for parasites: evidence supporting the North American invasion of European Echinococcus multilocularis. Proceedings of the Royal Society B: Biological Sciences, 290(1994), 20230128.

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