Genescan 500 liz size standard

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The GeneScan 500 LIZ size standard is a molecular weight marker used in capillary electrophoresis applications to determine the size of DNA fragments. It is a mixture of fluorescently labeled DNA fragments with known sizes that can be detected during electrophoresis.

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GeneScan 500 LIZ (101 citations) LIZ 500 (23 citations) GeneScan 500 (11 citations) Genescan 500 size standard (5 citations) GeneScanTM-500 LIZTM (3 citations) GeneScanTM 500 LIZTM size standard (3 citations)

156 protocols using genescan 500 liz size standard

STR-based iPSC Line Identification

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Six highly polymorphic tetranucleotide microsatellites (STRs) were amplified by PCR and genotyped using the Applied Biosystems™ 3730 DNA Analyzer (Thermo Fisher Scientific). Fragment sizes were measured against GeneScan™ 500 LIZ™ size standard (Themo Fisher Scientific) and allele sizes were analyzed using GeneMapper™ v 4.0 (Thermo Fisher Scientific). This identity screen was used to ensure the iPSC line matched the parental fibroblast line.

Mitchell M.W., Grandizio C., Turan N, & Requesens D.V. (2022). An induced pluripotent stem cell line (CIMRi001-A) from a Vici syndrome donor with a homozygous recessive c.1007A>G (p.Q336R) mutation in the EPG5 gene. Stem cell research, 63, 102833.

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STR-based iPSC Line Identification

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Grapevine Variety Identification via Microsatellite Analysis

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For variety identification and confirmation, eight vines were sampled. DNA was extracted using the peqGOLD Plant DNA mini kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer's instructions. Nine microsatellite (simple sequence repeats, SSR) primers recommended for routine variety distinction of grapevine were used (15 ). Polymerase chain reaction (PCR) amplifications were carried out in an Applied Biosystems Veriti™ thermal cycler (Thermo Fisher Scientific, Foster City, CA, USA). The list and information about the used primers, as well as detailed information on multiplex PCR reactions performed, are described by Žulj Mihaljević et al. (4 (link)). Amplified products were separated using an Applied Biosystems 3130 genetic analyzer (Thermo Fisher Scientific) with GeneScan™ 500 LIZ^® size standard. Sizing of the fragments was performed using GeneMapper v. 4.0 software (15 ). The obtained SSR profiles were compared to internal microsatellite database comprising profiles on 9 common loci from European Vitis database (16 ) as well as published SSR profiles from other research (4 (link)). Data were standardized and compared as described previously (4 (link)).

Pavlešić T., Saftić Martinović L., Peršurić Ž., Maletić E., Žulj Mihaljević M., Stupić D., Andabaka Ž., Grgić Z, & Kraljević Pavelić S. (2022). From the Autochthonous Grape Varieties of the Kastav Region (Croatia) to the Belica Wine. Food Technology and Biotechnology, 60(1), 11-20.

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Multiplex PCR for STR and CAG Repeats

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STRs were all amplified together with the CAG repeat, in a multiplex PCR reaction, in a final volume of 10 μL, using 0.25 μM (AAAC123, AC21, GT199, and GT190), 0.125 μM (TAT223, ATA194, and AC190), and 0.2 μM (CAG, and MJD52_F) of each primer, 1x of Taq PCR Master Mix Kit Qiagen^®, 0.5x of Q-Solution for Qiagen^®, and 7.5 ng DNA. The initial denaturation was performed at 95°C for 15 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 90 s and extension at 72°C for 60 s; with a final extension at 70°C for 30 min. Analysis of fragment length was performed with ABI PRISM 3130x/Genetic Analyzer (Applied Biosystems). A mix of GeneScan™ 500 LIZ™ size standard (Thermo Scientific):Hi-Di™ formamide (Applied Biosystems) (1:20) was added to 2 μL of PCR product and run in matrix G5, analyzed with GeneMapper v4.0. Allelic phases associated with the expansion were assessed by segregation and bioinformatically, using PHASE v.2.1.1 whenever DNA samples from relatives were not available.

Costa I.P., Almeida B.C., Sequeiros J., Amorim A, & Martins S. (2019). A Pipeline to Assess Disease-Associated Haplotypes in Repeat Expansion Disorders: The Example of MJD/SCA3 Locus. Frontiers in Genetics, 10, 38.

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Capillary Sequencing of FAM70 DNA Size

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Changes in size of the FAM70 DNA were analyzed by a capillary sequencing as described previously^{6 (link)}. Briefly, samples were added to the HiDi formamide-containing GeneScan–500 LIZ Size Standard (Thermo Fisher Scientific, USA), which contains 16 fragments of known size, and size-separated in a 3130xl Genetic Analyzer (Thermo Fisher Scientific, USA). TraceViewer software^{6 (link)} was used to analyze the data, in which it aligned different run data by using two peaks from the size standard.

Ohtsubo Y., Nagata Y, & Tsuda M. (2017). Compounds that enhance the tailing activity of Moloney murine leukemia virus reverse transcriptase. Scientific Reports, 7, 6520.

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Genomic DNA Extraction and Microsatellite Genotyping of Winter Moths

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Whole genomic DNA was extracted from individual male winter moths using the Qiagen DNeasy Kit (Qiagen Corporation, Netherlands) following the manufacturer's instructions. Prior to DNA extraction, the tip of the abdomen (genitalia are used for morphological species identification) and wings were removed and stored as vouchers for most of the samples. Vouchers were deposited at the Yale Peabody Museum of Natural History, New Haven Connecticut, USA. Twenty‐four polymorphic microsatellites were then amplified from each individual using primers and protocols presented in Havill et al. (2017). Genotyping was conducted at the DNA Analysis Facility on Science Hill at Yale University using a 3730xl DNA Analyzer (Thermo Fisher Scientific, USA), and fragment lengths were scored in comparison with the GeneScan 500 LIZ size standard (Thermo Fisher Scientific) using the microsatellite plug‐in in the software program geneious v.8.1.6 (Kearse et al., 2012).

Andersen J.C., Havill N.P., Caccone A, & Elkinton J.S. (2017). Postglacial recolonization shaped the genetic diversity of the winter moth (Operophtera brumata) in Europe. Ecology and Evolution, 7(10), 3312-3323.

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MLPA Analysis of DPYD Variants

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MLPA was performed using the SALSA MLPA probemix P103-C1 DPYD (MRC Holland), according to the manufacturer’s guidelines. This probemix contained 45 MLPA probes, including 33 probes for exons, two probes specific for the c.1129-5923C > G and the c.1905 + 1G > A (IVS14 + 1G > A) variants and two probes specific for the wild type sequence of the c.1679 T > G and the c.2846A > T variants. Additionally, nine quality control probes were included. The MLPA PCR products were run on an Applied Biosystems 3500XL Genetic analyzer using the GeneScan 500 LIZ Size Standard (Thermo Fisher Scientific) and the data were analysed with the Coffalyser software (MRC Holland).

Malekkou A., Tomazou M., Mavrikiou G., Dionysiou M., Georgiou T., Papaevripidou I., Alexandrou A., Sismani C., Drousiotou A., Grafakou O, & Petrou P.P. (2024). A novel large intragenic DPYD deletion causing dihydropyrimidine dehydrogenase deficiency: a case report. BMC Medical Genomics, 17, 78.

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Genotyping Microsatellites for Variant Analysis

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We were able to genotype 138 individuals from 44 unrelated European JS families, as well as 13 non-JS subjects, carrying the variants of interest. We selected 8–10 highly polymorphic microsatellites for each locus, spanning ~4 Mb around each variant (online supplemental table 1). Markers were PCR-amplified using fluorescent primers, mixed with formamide and GeneScan 500 LIZ Size Standard (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and run on 3500 Genetic Analyzer (Thermo Fisher Scientific). Raw data were analysed using GeneMapper Software V.6 (Thermo Fisher Scientific). Haplotypes were reconstructed by phasing alleles according to familial segregation. The haplotype around TMEM67 c.755T>C could not be analysed due to the lack of sufficient DNA samples.

Serpieri V., Mortarini G., Loucks H., Biagini T., Micalizzi A., Palmieri I., Dempsey J.C., D’Abrusco F., Mazzotta C., Battini R., Bertini E.S., Boltshauser E., Borgatti R., Brockmann K., D'Arrigo S., Nardocci N., Fischetto R., Agolini E., Novelli A., Romano A., Romaniello R., Stanzial F., Signorini S., Strisciuglio P., Gana S., Mazza T., Doherty D, & Valente E.M. (2023). Recurrent, founder and hypomorphic variants contribute to the genetic landscape of Joubert syndrome. Journal of Medical Genetics, 60(9), 885-893.

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Microsatellite Amplification and Analysis

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PCR were optimized to amplify all microsatellites carried out in an Heal Force Thermal Cycler Thermal T960 (Heal Force, Shanghai, China). Each PCR amplification was performed in a total volume of 25 µl containing 0.5 unit of Taq DNA polymerase and the PCR program was: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 60°C for 40 s, annealing temperature, and 72°C for 30 s, and a final extension at 72°C for 15 min. The PCR products were identified and classified by capillary electrophoresis using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Foster City, SA) and GeneScan 500 LIZ size standard (Thermo Fisher Scientific). Raw capillary electrophoresis data were analyzed using the Geneious v 8.0.5 software (Biomatters, http://www.geneious.com/).

Díaz-Matus de la Parra M., Inostroza K., Alcalde J.A., Larama G, & Bravo S. (2023). Characterization of the genetic diversity, structure, and admixture of 7 Chilean chicken breeds. Poultry Science, 103(2), 103238.

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ABCA7 Haplotyping via SNPs and STRs

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Haplotypes are based on 18 single nucleotide polymorphisms (SNPs) spanning ABCA7 and seven short tandem repeat (STR) markers flanking ABCA7 as described [10 (link)]. STR markers were PCR-amplified using fluorescently labelled primers. PCR-products were supplemented with GeneScan™ 500 LIZ™ size standard (Thermo Fisher Scientific, MA, USA) and size-separated using capillary electrophoresis on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific, MA, USA). STR fragment lengths were assigned using the Local Genotype Viewer software (https://www.neuromicssupportfacility.be/) developed by the Neuromics Support Facility, Center for Molecular Neurology, Antwerp, Belgium.

Bossaerts L., Hendrickx Van de Craen E., Cacace R., Asselbergh B, & Van Broeckhoven C. (2022). Rare missense mutations in ABCA7 might increase Alzheimer’s disease risk by plasma membrane exclusion. Acta Neuropathologica Communications, 10, 43.

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