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[…] al volume of 50 μL. The genomic PCR conditions included: 95 °C for 3 min followed by 35 cycles at 95 °C for 15 s and 46.9 °C (52.0, 56.2, 56.2, 58.6 °C) for 15 s, 72 °C for 2 min and a final extension at 72 °C for 5 min. The products were analyzed by 1% agarose gel electrophoresis., PCR products of DKK1 cDNA and genomic fragments were purified using the Agarose Gel Extraction Kit according to the manufacturer’s instructions (TIANGEN, Beijing, China) and cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA). The recombinant plasmids were extracted using PureLinkR Quick Plasmid Miniprep kit (Invitrogen) and sequenced by Invitrogen. The sequences were aligned using the Align X function of Vector NTI program (Informax, Rockville, MD, USA). The homologous DKK1 mRNA sequences of 16 different animal species used in this study were obtained from National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) database (). Homology analysis was performed using the Align Sequences Nucleotide BLAST utility at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Translation of the nucleotide sequences into the amino acids was performed using the DNAMAN program (Lynnon Corp., Quebec, Canada). Exon–intron boundaries were identified by alignment of the acquired Merino DKK1 genomic DNA sequence (GenBank accession No. JQ348893.1) and the acquired Merino DKK1 cDNA sequence using the Align Sequences Nucleotide BLAST at NCBI. The signal peptide was analyzed using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). The conserved domains were analyzed using a conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/cdd/). Transcription factor binding sites (TFBS) were predicted using Mulan (https://mulan.dcode.org/). Core promoter was predicted using Promoter SCAN (http://www-bimas.cit.nih.gov/molbio/proscan/). The neighbor-joining phylogenetic tree was constructed using the Phydit program version 3.0 [] based on genetic distances calculated with Kimura’s two-parameter method []., To screen for DKK1 polymorphisms, a total of five primer pairs (DKK1-F2/R2-DKK1-F6/R6) were used to amplify DKK1 genomic region () using the pooled gDNA as the templat […]

Pipeline specifications

Software tools Vector NTI Advance, DNAMAN, SignalP
Databases CDD
Organisms Ovis aries
Diseases Sprains and Strains, Wiskott-Aldrich Syndrome
Chemicals Amino Acids