Genotyping of Candidate SNPs and Variants

Genotyping of the candidate SNPs LEPREL1:g.139212C>G, KRT3:g.2584C>T as well as the polydactyly-associated polymorphism in LMBR1 (DC-2) was performed using restriction fragment length polymorphisms according to standard protocols [52 (link)]. DC-2 primers were obtained from previous study [18 ]. In addition the missense variants CEP164:g.57380G>T and COL28A1:g.159951T>A were validated using Kompetitive Allele Specific PCR (KASP) genotyping assays (LGC, Middlesex, UK) [53 ]. After the KASP standard thermal cycling touchdown protocol was run on a thermocycler TProfessional 96 (Biometra, Göttingen, Germany) using an annealing temperature of 61 °C (Additional file 12), allelic discrimination was performed on the ABI7300 sequence detection system (Applied Biosystems, Waltham, Massachusetts, USA).

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Metzger J., Pfahler S, & Distl O. (2016). Variant detection and runs of homozygosity in next generation sequencing data elucidate the genetic background of Lundehund syndrome. BMC Genomics, 17, 535.

Publication 2016

TProfessional 96 ABI7300 sequence detection system

Allelic Assays Discrimination Missense variants Polydactyly Polymorphism Primers Restriction fragment length polymorphisms Snps

Corresponding Organization : University of Veterinary Medicine Hannover, Foundation

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Variable analysis

independent variables

Genotyping of the candidate SNPs LEPREL1:g.139212C>G, KRT3:g.2584C>T, and the polydactyly-associated polymorphism in LMBR1 (DC-2)
Validation of the missense variants CEP164:g.57380G>T and COL28A1:g.159951T>A using Kompetitive Allele Specific PCR (KASP) genotyping assays

dependent variables

Allelic discrimination performed on the ABI7300 sequence detection system

control variables

Restriction fragment length polymorphisms according to standard protocols [52]
KASP standard thermal cycling touchdown protocol run on a thermocycler TProfessional 96 using an annealing temperature of 61 °C

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