Untersuchungen zu familiären und rassespezifischen ...

Genotyping and sequence analysis
Genomic DNA was isolated from EDTA blood samples using the QIAamp® 96 Spin
Blood Kit (Qiagen, Hilden, Germany). For genotyping microsatellites, forward primers
were labeled fluorescently at the 5’-end with IRD700 or IRD800. PCR-amplicons
were fractionated according to their length by polyacrylamide electrophoresis in 6%
gels using the automated sequencer LI-COR 4200/S-2 (LI-COR, Lincoln, NE, USA).
Size determination was performed by visual comparison to an IRD700- and IRD800-
fluorescence-labeled DNA ladder.
For scanning of the SNPs from the Broad Institute SNP collection, the PCR-
amplicons of four GE-affected and four non-affected Norwegian lundehunds were
sequenced using the MegaBACE 1000 automated sequencer (GE Healthcare,
Freiburg, Germany). Sequence data were analyzed using the Sequencher 4.7
program (GeneCodes, Ann Arbor, MI, USA).
Primers were designed for all seven coding exons of the AMN gene annotated at the
NCBI database (GeneID: 403434). These primer sequences for the genomic DNA of
the AMN gene are shown in Table 3. PCR was performed using the ThermalAce
DNA Polymerase kit (Invitrogen) following the manufacturer’s specifications. The
same four GE-affected and four non-affected animals were chosen for sequencing
these coding regions.
Data analysis
Mendelian inheritance and correctness of marker transmission in the genotyped dogs
was confirmed using the Pedstats software (Wigginton and Abecasis, 2005).
Multipoint non-parametric linkage analysis (NPL) was performed using the MERLIN