Computational protocol: Loss or major reduction of umami taste sensation in pinnipeds

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Protocol publication

[…] Partial DNA sequences of Tas1r1 exon 3 were newly determined for six pinnipeds and six other carnivorans. These sequences have been deposited in the DDBJ/EMBL/GenBank databases with accession numbers AB697513–AB697524. Homologous sequences for two other carnivorans, a polar bear (HM468451) and a domestic dog (HM468447), were obtained from the DDBJ/EMBL/GenBank databases (Fig. ).Fig. 1Total genomic DNA was extracted from either tissue or blood using a standard phenol–chloroform method or a DNeasy Blood and Tissue Kit (Qiagen). Amplification was performed through PCR reactions with a KOD-plus-Neo DNA polymerase kit (Toyobo) in an automated thermal cycler (model PC 302, Astec). Each PCR mix contained KOD-plus-NEO buffer, 1.5 mM MgSO4, 0.2 mM of each dNTP, 0.3 μM of each of Tas1r1_ex3_Fw1 (5′-GGAGTGAAGCGGTATTACCC-3′) and Tas1r1_ex3_Rv1 (5′-GGCCTCTTCAAACTCCTTCAGGC-3′) primers (both newly designed), 1.0 U of KOD-Plus-Neo DNA polymerase, and 0.1–0.2 μg of template total genomic DNA in a total volume of 50 μl. The PCR thermal cycling parameters included a 2-min denaturation period at 94 °C followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and extension at 68 °C for 30 s; this was followed by a 10-min extension period at 68 °C. Sequencing reaction was carried out with a Big Dye Terminator (v. 3.1) Cycle Sequencing Kit (Applied Biosystems). Sequences were resolved using an ABI3130 automated sequencer (Applied Biosystems).Multiple sequence alignment was accomplished in MEGA 5 (Tamura et al. ). Cladistic analysis was conducted in GARLI 2.0 (Zwickl ) using maximum likelihood with the HKY + Γ model of DNA substitution. This optimal model was identified with the Akaike information criterion implemented in Modeltest 3.7 (Posada and Crandall ). Insertions were not considered. Deletions and nonsense substitutions were coded as missing data. Both canids (domestic dog and red fox) were used as an outgroup. Tree searching was heuristic. Five runs of the genetic algorithm were performed, each with 50,000 generations of a mutation–selection–reproduction cycle. Starting trees were generated through stepwise addition. Bootstrap percentages were computed from 1,000 pseudoreplicates. […]

Pipeline specifications

Software tools MEGA, GARLI, ModelTest-NG
Databases DDBJ
Application Phylogenetics
Organisms Homo sapiens
Chemicals Sodium Chloride