L dna sequencer

For full-length sequencing of HBV genome, we used the same primers used for PCR in the previously described method^{21 (link),36 (link)}. Briefly, we extracted 100 µL of serum for detection of HBV DNA using SMITEST EX-R&D (Genome Science Laboratories, Fukushima, Japan) and for nested PCR using Prime STAR^®GXL polymerase (Takara Bio Inc., Shiga, Japan) with the primer set WA-L and WA-R and inner primers WA-L2 and WA-R2. For the missing portion of the circular HBV DNA, we performed nested PCR again on the extracted DNA using Prime STAR^®GXL polymerase and the primer sets S1, S2, AS1, and AS2. Final products were sequenced using an Applied Biosystems 3730 x l DNA sequencer (Thermo Fisher Scientific K.K., Kanagawa, Japan) and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

For full-length genome sequences, the same primers as of the previously described method were used in this study [16 (link), 17 ]. The amplification was carried out by nested polymerase chain reaction (Nested-PCR) using Prime STAR^®GXL polymerase (Takara Bio Inc., Shiga, Japan) and the primer set A (WA-L and WA-R and inner primers WA-L2 and WA-R2) [16 (link)]. For the missing portion of the circular HBV DNA, the extracted DNA was assigned again for the nested PCR using Prime STAR^®GXL polymerase (Takara Bio Inc., Shiga, Japan) and the primer set B (S1, S2, AS1, and AS2). The obtained PCR product was directly sequenced using a 3730xl DNA sequencer (Thermo Fisher Scientific K.K., Kanagawa, Japan) and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
The samples which were not detected by WA primer set, were then attempted for s-region fragment (partial genome sequence) using the primer set #S1–1 and #S1–2 and the inner primers #S2–1 and #S2–2 [18 (link), 19 (link)]. The obtained PCR products were directly sequenced as the same way mentioned in full length sequences.