OLIGO 7 Primer Analysis Software

“…Agarose gel electrophoresis of the mtDNA sample with the total DNA showed no contamination from the chromosomal DNA. Based on the complete mtDNA sequences of F. catus (NC001700), A. jubatus (AY463959), and N. nebulosa (DQ257669), and some partial sequences of P. tigris (DQ151550), we successfully designed 34 pairs of primers for amplifying the complete mitochondrial genome sequences of the three species using Oligo 6.0 [21] (Table 1). PCR reactions were performed in an MJ Model PTC-200 thermal cycler with the following conditions: 95C for 5 min; 30 cycles of 94C for 50 s, 52C-56C for 1 min, 72C for 1 min; and 72C for 10 min.…”

Section: Samples Sources Dna Extraction and Pcr Amplificationmentioning

confidence: 99%

Mitogenomic analysis of the genus Panthera

Wei

Xiao-bing

Zhu

et al. 2011

Sci. China Life Sci.

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The complete sequences of the mitochondrial DNA genomes of Panthera tigris, Panthera pardus, and Panthera uncia were determined using the polymerase chain reaction method. The lengths of the complete mitochondrial DNA sequences of the three species were 16990, 16964, and 16773 bp, respectively. Each of the three mitochondrial DNA genomes included 13 protein-coding genes, 22 tRNA, two rRNA, one O L R, and one control region. The structures of the genomes were highly similar to those of Felis catus, Acinonyx jubatus, and Neofelis nebulosa. The phylogenies of the genus Panthera were inferred from two combined mitochondrial sequence data sets and the complete mitochondrial genome sequences, by MP (maximum parsimony), ML (maximum likelihood), and Bayesian analysis. The results showed that Panthera was composed of Panthera leo, P. uncia, P. pardus, Panthera onca, P. tigris, and N. nebulosa, which was included as the most basal member. The phylogeny within Panthera genus was N. nebulosa (P. tigris (P. onca (P. pardus, (P. leo, P. uncia)))). The divergence times for Panthera genus were estimated based on the ML branch lengths and four well-established calibration points. The results showed that at about 11.3 MYA, the Panthera genus separated from other felid species and then evolved into the several species of the genus. In detail, N. nebulosa was estimated to be founded about 8.66 MYA, P. tigris about 6.55 MYA, P. uncia about 4.63 MYA, and P. pardus about 4.35 MYA. All these estimated times were older than those estimated from the fossil records. The divergence event, evolutionary process, speciation, and distribution pattern of P. uncia, a species endemic to the central Asia with core habitats on the Qinghai-Tibetan Plateau and surrounding highlands, mostly correlated with the geological tectonic events and intensive climate shifts that happened at 8, 3.6, 2.5, and 1.7 MYA on the plateau during the late Cenozoic period.

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“…Experiment I: confirming the presence of specific sequences in genomic DNA To test whether the different combinations of flanking sequences were an artifact caused by enrichment procedures, or in fact occur in the observed association in the butterfly genome, we designed primers with OLIGO version 6 (Rychlik, 2000) to amplify 15 different combinations of symmetrical and asymmetrical sequence clusters in the ATG library. Product was detected with ethidium bromide-stained 1% agarose gel.…”

Section: Detection Of Inter-specific Homologiesmentioning

confidence: 99%