Bigdye terminator v3.1 kit

PCR was performed using an iTERT Mutation Detection Kit (GENINUS Inc., Seoul, Korea), according to the manufacturer's instructions. The PCR reactions were assembled on ice and preincubated at 94 °C for 15 min, followed by 40 cycles at 94 °C for 20 s, 58 °C for 40 s, 72 °C for 1 min, and a final extension at 72 °C for 5 min using a C1000 Touch Thermal Cycler Kit (Bio-Rad, Hercules, CA). Bidirectional sequencing was performed using the BigDye Terminator v.3.1 Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130xL Genetic Analyzer. The results were marked as mutation-positive if a mutation was detected in both the forward and reverse DNA strands [31 (link)]. Positive controls were included in each sequencing run: normal human guide DNA (gDNA) (wild-type) and cancer cell (e.g., the C228T‐positive MDA-MG-231 cell line)-derived genomic DNA that yielded the expected TERT promoter sequences in each case.

Point mutations and polymorphisms can disrupt the hybridization site of the MLPA probes and produce a false deletion in the quantitative analysis. For this reason, samples that showed deletions in the MLPA assay were submitted to sequencing. The reaction was prepared according to the manufacturer's protocol using the Big Dye Terminator v3.1 Kit (Applied Biosystems, Foster City, CA). All reactions were processed on the ABI Prism 3130 automatic sequencer (Applied Biosystems, Foster City, CA) in the DNA Sequencing Platform at Oswaldo Cruz Foundation/ FIOCRUZ, Brazil. Sequence analysis was performed with Chromas Lite 2.0 software (Technelysium) and BioEdit Sequence Alignment Editor v6.0.6 (Isis Pharmaceuticals, Inc.).

Amplicons were analyzed using agarose gel electrophoresis. Depending on the
presence of a single or multiple bands on the gel, amplicons were either
directly purified with QIAquick PCR Purification kit or gel-isolated and
purified using QIAquick Gel Extraction kit (Qiagen, Courtaboeuf, France)
following the manufacturer’s protocol.
Resulting amplicons were subjected to direct sequencing using the BigDye
terminator v3.1 kit (Applied Biosystems) and an ABI Prism 3140 automated
sequencer (Applied Biosystems). The sequencing of each amplicon was performed in
both directions using semi-nested PCR primers. Sequence data obtained using the
ABI PRISM kits were viewed, assembled and edited using the CLC Main Workbench
5.7.2 software (CLC bio, Aarhus, Denmark). Consensus sequences were submitted to
databases with the following accession numbers: MK310554 to MK310568 and
MK310788 to MK310984 respectively for the VP1 and VP3-VP1 sequences of NPEVs;
MK310569 to K310641, MK310642 to MK310714 and MK310715 to MK310787 respectively
for the 2C^ATPase, 3D^pol and 5’UTR sequences of cVDPVs;
MK310985 to MK311038 for the full-length VP1 sequences of cVDPVs; MK311039 to
MK311132 for the full-length VP1 sequences of Sabin-like PVs.