Computational protocol: A Mutation in Myo15 Leads to Usher-Like Symptoms in LEW/Ztm-ci2 Rats

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Protocol publication

[…] Resequencing of all exons of Myo15 and of Kcnj12 was performed using LIMSTILL, LIMS for Induced Mutations by Sequencing and TILLing ( LIMSTILL was used to generate the Myo15 project and visualize the gene structure based on Ensembl file ENSRNOG00000028597. The primer design application within LIMSTILL is Primer3-based and parameters are set to design primers with an optimal melting temperature of 58°C.PCR was performed using a touchdown thermocycling program (92°C for 60 sec; 12 cycles of 92°C for 20 sec, 65°C for 20 sec with a decrement of 0.4°C per cycle, 72°C for 30 sec; followed by 20 cycles of 92°C for 20 sec, 58°C for 20 sec and 72°C for 30 sec; 72°C for 180 sec; GeneAmp9700, Applied Biosystems). PCR reaction mixes contained 5 µl genomic DNA, 0.2 µM forward primer and 0.2 µM reverse primer, 200 µM of each dNTP, 25 mM Tricine, 7.0% Glycerol (w/v), 1.6% DMSO (w/v), 2 mM MgCl2, 85 mM Ammonium acetate pH 8.7 and 0.2 U Taq Polymerase in a total volume of 10 µl.PCR products were diluted with 20 µl water and 1 µl was used as template for the sequencing reactions. Sequencing reactions, containing 0.25 µl BigDYE (v1.1; Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), 3.75 µl 2.5× dilution buffer (Applied Biosystems) and 0.4 µM gene specific primer in a total volume of 10 µl, were performed using cycling conditions recommended by the manufacturer. Sequencing products were purified by ethanol precipitation in the presence of 40 mM sodium-acetate and analyzed on a 96-capillary 3730XL DNA analyzer (Applied Biosystems). Sequences were analyzed using PolyPhred . […]

Pipeline specifications

Software tools Primer3, PolyPhred
Applications Sanger sequencing, qPCR
Organisms Rattus norvegicus, Homo sapiens
Diseases Movement Disorders, Retinal Degeneration, Retinal Diseases, Genetic Diseases, Inborn