Verification of Treponemal Protein Genes via Nested PCR

To verify the RFLP patterns observed following restriction digestion, we used a nested PCR to amplify the full-length tpr genes separately for sequencing analysis[14 (link)]. Two μL of DNA was amplified in a 50 μL reaction with 2.5 units of TaKaRa polymerase (TaKaRa Bio, Inc., Mountain View, CA, USA), 1x PCR buffer, 200 nM dNTPs, and 0.2 μM of primers E1 and E2 for tpr E, G9.89 and G10.3026 for tpr G, or J2 and J3 for tpr J. Reaction conditions were as follows: 94°C for 1 min, then 35 cycles of 94°C for 30 sec, 60°C for 1 min, and 68°C for 3 min 30 sec, followed by a final extension at 68°C for 10 min. Two μL of outer PCR product were used for the nested reaction, which was carried out using the same conditions but with primers E3 and E4 for tpr E, G6.232 and TPRG8 for tpr G, and TPRJ9Fl and TPRJ11 for tpr J. Samples were run on an Agilent Bioanalyzer and subsequently purified using the QIAquick PCR Purification Kit for amplicon sequencing.
To verify the number of 60-bp repeats as well as the sequence of each repeat within arp, we purified all arp amplicons using the QIAquick PCR Purification Kit.