Amplification and Sequencing of Ca. L. asiaticus Loci

Four loci within the ‘Ca. L. asiaticus’ genome containing TACAGAA, CAGT, AGACACA, and TTTG motifs [20 (link), 21 (link), 27 (link)] were examined in this study. These motifs had been previously designated as 001, 002, 005, and 077, respectively, [21 (link)]. The fragments containing these loci were amplified by PCR using the primer sets listed in Table 1, as previously reported [21 (link)]. Additionally, another forward primer was designed in order to be easy to count the motif of VNTR ‘001’ by using a program available on the Primer3 website (http://frodo.wi.mit.edu/primer3/) (Table 1).
PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) in a 20-μL reaction mixture containing 1 μL of DNA template, 0.1 μM of each primer, 200 μM dNTP mixture, 1× PCR buffer, and 2.5 units of Ex Taq DNA polymerase Hot Start Version (TaKaRa, Shiga, Japan). The thermal cycling conditions were as follows: initial denaturation at 92°C for 2 min; 35 cycles of denaturing at 92°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min. Amplified PCR products were separated by electrophoresis in a 1.5% (wt/vol) agarose gel in Tris-boric acid EDTA buffer. Before direct sequencing, the PCR products were extracted from the gel slices using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions.