Sanger Sequencing of Genetic Variants

Primers were designed using the Primer3 online software^{42 (link)}, at least 70 bp upstream and downstream of the variants, and PCR reactions were performed under standard conditions (for the list of primers see Supplementary Table 4). PCR products were purified by treatment with exonuclease and thermosensitive alkaline phosphatase (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed by Sanger sequencing using the Big Dye Terminator v3.1 Cycling Sequencing Kit (Applied Biosystem, Foster City, CA, USA) on an ABI 3730XL platform (Applied Biosystems). Sequencing data were analyzed using the Sequencher v4.8, SnapGene (https://www.snapgene.com), and Chromas Lite v2.01 software and compared with wild-type samples and reference sequences from NCBI and Ensembl databases. Furthermore, frequency of all candidate variants was verified in a database of 800 Iranian healthy individuals (https://www.iranome.ir/).

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Salmaninejad A., Bedoni N., Ravesh Z., Quinodoz M., Shoeibi N., Mojarrad M., Pasdar A, & Rivolta C. (2020). Whole exome sequencing and homozygosity mapping reveals genetic defects in consanguineous Iranian families with inherited retinal dystrophies. Scientific Reports, 10, 19413.

Publication 2020

exonuclease and thermosensitive alkaline phosphatase ABI 3730XL platform

Alkaline phosphatase Exonuclease Primers

Corresponding Organization :

Other organizations : Mashhad University of Medical Sciences, University Hospital of Lausanne, University of Leicester

Top 5 similar protocols

Variable analysis

independent variables

Primer design using Primer3 online software
PCR reaction conditions

dependent variables

Sanger sequencing of PCR products
Sequence data analysis using software (Sequencher, SnapGene, Chromas Lite)

control variables

Comparison with wild-type samples and reference sequences from NCBI and Ensembl databases
Verification of variant frequency in a database of 800 Iranian healthy individuals

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