BMC Cancer 2005, 5:102 http://www.biomedcentral.com/1471-2407/5/102 Table 1: Primers used for amplification of Mus Musculus mitochondrial genome Primer pair mtDNA bp Forward primer Reverse primer 1 2 3 4 5 6 7 8 9 10 11 12 15309-00040 16173-01436 01314–02880 02673–03873 03731–05324 05194–06593 06477–07996 07847–09438 09307–10806 10647–12399 12247–13788 13670–15498 GGTCTTGTAAACCTGAAATGAAG CTGATCAATTCTAGTAGTTCC GAATTACAGCTAGAAACCCCG GTTATTAGGGTGGCAGAGCCAG CTTTGATAGAGTAAATTATAGAGG GTCTTAGTAGAGATTTCTCTACAC GGAGCAGTGTTTGCTATCATAGC GTCTCATCACAAACATTCCCAC GATTTGAAGCCGCAGCATGATAC GCCCTCATCTTAATCCAAAACC CAATCCTCTATAACCGCATCG CCAACATCATCAACCTCATAC GCATTTTCAGTGCTTTGCTTTG CAAGCTCGTTAGGCTTTTCAC CGTATGGACCAACAATGTTAGG GCCCGATAGCTTAATTAGCTGAC TAAAATGGCTGAGTAAGCATTAG GTTTACTCCTACGAATATGATGG TGATGGCTACAACGATTGGGAATC CAGAATCTACTAATTGGAAGTCAG GTCATAGGTGAACTCCATATAATGG CTGTAGCTGCGATTAATAGGC GTATTGGGGGTGATTATAGAG GTACTAGCTTATATGCTTGGG The nucleotide positions were based on Mus Musculus sequence from Bibb et al. 1981 [GenBank: V00711] Table 2: Summary of mtDNA nucleotide variations in C57BL/6J and VM mouse strainsa Mouse strain Nucleotide position DNA change Amino acid change Gene ND5 ND5 Arg tRNA ND3 C57BL/6J VM VM VM 11780 11780 9829 9461 T->A T->A InsA T->C Ile->Met Ile->Met NAb Ile->Ile Accession numbers for C57BL/6J and VM strains are DQ106412 and DQ106413, respectively a Nucleotide variation compared to the republished C57BL/6J mtDNA sequence from Bayona-Bafaluy et al. 2003 b NA, not applicable CA, USA) and was amplified in 12 overlapping segments covering the entire mitochondrial genome. PCR reactions were performed in a volume of 50 µ l using the Eppendorf Master Taq system (Brinkmann Eppendorf, Westbury, NY). Primers used for mtDNA amplification are given in Table 1. PCR conditions for all primer pairs consisted of an initial denaturation at 94°C for 120 s followed by 34 cycles of 94°C for 60 s, an annealing temperature of 57– 66°C for 35 s and 72°C for extension at 105 s. A final extension was for 5 min. Optimal annealing temperatures were determined by performing gradient PCR on each of the primer sets. mtDNA sequencing Amplified regions of mtDNA were purified using a PCR purification kit (MO BIO Laboratories, Solana Beach, CA) and were then visualized by 1% agarose gel electrophore- sis. DNA concentration was estimated using a Low DNA Mass Ladder (Invitrogen, Carlsbad, CA) by gel electro- phoresis. Approximately 25 fmol of the purified PCR product was used in the sequencing reaction following manufacturer's instructions for the CEQ DTCS kit (Beck- man Coulter, Fullerton, CA). The amount of DNA used varied depending on the size of the PCR fragment. The primers in Table 1, together with 75 nested primers, were used in the sequencing reactions to obtain double- stranded sequence. Nested primer information can be made available upon request to corresponding investigators. Sequencing reactions were performed at 96°C for 20 s, 50°C for 20 s, and 60°C for 240 s based on a 32 cycle reac- tion. Sequencing products were ethanol precipitated in cold 95% ethanol, washed twice with cold 70% ethanol, dried for 20 minutes, and then re-dissolved in sample loading solution provided by the manufacturer (Beckman Coulter, Fullerton, CA). The mtDNA sequence alignments were then compared to those from Mus Musculus [Gen- Bank: V00711] [56] and from B6 mice [GenBank: AY172335] [23]. Results Analysis of mtDNA variation in B6 and VM mouse strains Sequence analysis of the entire mitochondrial genome of the VM and B6 mouse strains revealed a single nucleotide base pair change at position 11780, found in both strains, compared to the republished B6 sequence [23]. This resulted in a T->A transversion, causing an amino acid Page 3 of 8 (page number not for citation purposes)