Long-amp PCR based Sanger sequencing for Sphenodon mitochondrial genome
Genomic DNA was extracted from liver of two female Tuatara previously residing at the St. Louis Zoo [BioSample SAMN10598679 (ISIS379002, SLZ 14846) and BioSample SAMN10598680 (ISIS103796, SLZ 19079)] originating from Stephens Island, Marlborough District, New Zealand, using the Qiagen Genomic Tip DNA extraction kit (genomic-tip column 100/G) from ~20mg of liver tissue according to the manufacturer's directions at the Laboratories of Analytical Biology at the Smithsonian Institutions National Museum of Natural History (NMNH). Two sets of forward and reverse primers were designed on the Stephens Island (BioSample SAMN10598677, ISIS 373002) Sanger sequences generated from Long-amp PCR. Two forward (Rex26_ND5F1) 5-GTGCACTAACACAAAACGATATC-3 and (Rex27_ND5F1) 5-GCGCACTGACACAAAATGATATT-3 and two reverse primers (Rex26_ND5R1) 5-GGATTCCTCCTATTTTTCGAATG-3 and (Rex27_ND5R1) 5-GGATTCCTCCTATTTTTCAGATA-3 were designed in the middle of the ND5 gene, where the two mt-genome copies differed significantly. Primer sets were designed to amplify only one or the other of the differing copies (Rex26Rex27). Forward ND5 primers were used with the 12S rRNA primer (H1067; Macey et al. 1997, Mol. Biol. Evol., 14, 91104) 5-TAGTGGGGTATCTAATCCCAGTTT-3 and reverse ND5 primers were used with the COIII primer (L9940) 5-GCAGCATGATACTGACACTTYGT-3 (this study) in long-amp reactions, producing fragments ~5 kb long. Polymerase chain reactions (PCR) were conducted on the two separate [BioSample SAMN10598679 (ISIS379002, SLZ 14846) and BioSample SAMN10598680 (ISIS103796, SLZ 19079)] Qiagen Genomic Tip DNA extractions, using LongAmp taq (New England BioLabs Inc. #M0323S), in 25 ul reactions, with 12 ul of template, and amplified with a beginning denaturation at 94 C for 45 s, then followed by 37 cycles of a denaturation at 94 C for 15 s, annealing at 50 C for 20 s, and extension at 68 C for 9 min, with a final extension at 72 C for 12 min after the last cycle. Negative controls were run on all amplifications to check for contamination. PCR products were purified using ExoSapIT and end-sequence reactions were performed with the PCR primers using BigDye Terminator v3.1 Cycle Sequencing Kits in 0.25  10 ul reactions and run on an Automated ABI3730 Sequencer (2011 Life Technologies) with the 900 chemistry. Additionally, internal primers were also used in cycle-sequence reactions on the long-amp (~5 kb) products, including the forward primer (ND4) 5-CACCTATGACTACCAGCTCATGTAGAAGC-3 (Arevelo et al. 1994, Systematic Biology 43:387418) and the reverse primer (IguaCytoBR2) 5- GGTTTACAAGACCAATGCTTT-3 (Corl et al., 2010, PNAS 107, 42544259). Using Geneious v10.2.4 (Biomatters Ltd 2005-2017), raw trace files from both sample extractions were initially aligned with the Lady Alice first molecule mitochondrial genome (BioSample SAMN10598677, ISIS 373002), and the BioSample SAMN10598680 (ISIS103796, SLZ 19079) sequences were later aligned with the PacBio-produced mitochondrial genome of the same individual resolving several ambiguities. Trace files are named by the specimen number (SLZ 14846 and SLZ 19079) followed by Spheno_Rex26 or Rex27 (from which fragment ND5f or ND5r primers were amplified and where f designates light-strand extending and r designates heavy-strand extending primers), followed by the primer used for sequencing.