Appendix 1. Protocol used for PCR amplification and DNA sequencing
Twenty-five overlapping fragments of the mitochondrial genome were amplified by
PCR using primers published in Hassanin et al. (2009) and Hassanin et al. (2012), as well as
several new primers specially designed for this study: U400M1 (5’-CGA GCT TAA TCA
CCA WGC CKC GDG AAA-3’) / 12SL600 (5’-GGT ATC TAA TCC CAG TTT G-3’);
12SU1230 / 12SL2226M1LA; 12SU829 / 16SL518; 16SU365 / 16SL1056; 16SU946 /
N1L64; U16S1421 / LMet2; N1U840 / N2L492; N2U354M2 / AsnL; TrpUM1 (5’-CYA
GAC CAA RGR CCT TCA AAG-3’) / C1L705; UTyr / C1L1017; C1U897M1 / C2L15M1;
SerUM1 / A8L1; C2U603 / C3L45; A6U654M2 / GlyLM1; C3U420CA (5’-GGA GTM
TCM ATY ACC TGA GCC CA-3’) / LArgLA (5’-AAT CTA ATG AGT CGA AAT CA-3’);
C3U780M1 / L366; UArg / N4L918M1; N4U681 / Leu2L; Ser2U / N5L652; N5U501 /
N5L1214; N5U1146M5 / N5L154M5; N6RU102 / CBL402; GluMA / ProMA (5’-AGA
AYW TCA GCT TTG GGD GYT GAH GGT G-3’); U844 / L482; and UThr13 (5’-TAA
ART GAA GAG TCY HHG TAG TA-3’) / L482. Amplifications were done in 20 μl using 3
μl of Buffer 10X with MgCl2, 2 μl of dNTP (6.6 mM), 0.12 μl of Taq DNA polymerase (2.5
U, Qiagen, Hilden, Germany) and 0.75 μl of the two primers at 10 μM. The standard PCR
conditions were as follows: 4 min at 94°C; 5 cycles of denaturation / annealing / extension
with 45s at 94°C, 1 min at 60°C and 1 min at 72°C, followed by 30 cycles of 30s at 94°C, 45s
at 55°C, and 1 min at 72°C, followed by 10 min at 72°C. Both strands of PCR products were
sequenced using Sanger sequencing on an ABI 3730 automatic sequencer at the Centre
National de Séquençage (Genoscope) in Evry (France). The sequences were edited and
assembled using Sequencher 5.1 (Gene Codes, Corp., Ann Arbor, MI, USA).
References
Hassanin A, Delsuc F, Ropiquet A, Hammer C, Jansen van Vuuren B, Matthee C, RuizGarcia M, Catzeflis F, Areskoug V, Nguyen TT, Couloux A. 2012. Pattern and timing
of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a
comprehensive analysis of mitochondrial genomes. C R Biol. 335:32-50.
Hassanin A, Ropiquet A, Couloux A, Cruaud C. 2009. Evolution of the mitochondrial
genome in mammals living at high altitude: new insights from a study of the tribe
Caprini (Bovidae, Antilopinae). J Mol Evol 68:293-310.