Computational protocol: RUNX2 tandem repeats and the evolution of facial length in placental mammals

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Protocol publication

[…] RUNX2 DNA sequence data were obtained using both online genomic resources (24 species), and DNA isolation, amplification and sequencing (17 species). We retrieved DNA sequences corresponding to the poly-glutamine (polyQ) and poly-alanine (polyA) tandem repeat and flanking regions of RUNX2 from Ensembl (releases 63 and 64; 17 species) and Genbank via BLASTN [] (7 species) (Table ). Notably, the QA ratio could not be reconstructed from the fragmented reads of the Neanderthal genome [].For the 17 species sequenced de novo in this study (Table ), samples came from live animals and museum specimens, and sampling complied with institutional animal care and use protocols. Samples from live animals included feces, as well as hair collected during routine handling (e.g. for health checks). Museum specimens included skin, muscle, connective tissue, and bone. All xenarthran samples come from the Collection of Preserved Mammalian Tissues of the Institut des Sciences de l'Evolution, Montpellier [].We extracted DNA from approximately 0.02 to 0.05g of each sample tissue using the QIAamp DNA Mini kit (Qiagen) following the manufacturer’s instructions. PCR primer sequences are the same as those used by Sears et al. (2007). PCRs were performed using the Extensor Hi-Fidelity PCR enzyme (Thermo Scientific). An external touchdown PCR using primers Sears Ext F (5’-TTGTGATGCGTATTCCCGTA-3’) or Sears Int F (5’ATCCGAGCACCAGCCGGCGGCGCTTCAG-5’) with Sears Ext R (5’-ACSGAGCACAGGAAGTTGGG-3’) was performed on approximately 100ng of template DNA with the following cycling conditions: 95°C 2mins, 15x (95°C 30s, 61.3 (down 0.5°C per cycle) 30s, 72°C 50s), 20x (95°C 30s, 54.3°C 30s, 72°C 50s), 72°C 5 mins final extension. The product of the external PCR was then diluted to a 1/10 concentration and 0.5μl was added as template for the internal PCR. This nested PCR using primers Sears Int F and Sears Int R (5’-GTGGTCVGCGATGATCTCSAC-3’) was run with the following cycling condition: 94°C 3 mins, 40x (94°C 30s, 62°C 45s, 72°C 45s), 72°C 5 mins final extension. The product of this second reaction was run out on a 1.5% agarose gel containing ethidium bromide. When an amplicon of roughly 300bp in size was visible under UV light, the DNA was extracted, purified (QIAquick PCR purification kit, Qiagen) and sequenced (Big Dye, version 1.3).The RUNX2 DNA sequences were analyzed and translated using sequence alignment and editing software (Lasergene, DNASTAR, BioEdit, ClustalW) and the QA ratios were calculated from the translated sequences. Sequences of sufficient length (> 100bp) have been deposited in GenBank (accession numbers: JQ405327 - JQ405335). […]

Pipeline specifications

Software tools BLASTN, MUSCLE, BioEdit, Clustal W
Databases PolyQ
Application Nucleotide sequence alignment
Organisms Canis lupus familiaris, Homo sapiens
Diseases Facial Dermatoses