Molecular Cloning of TMPRSS2-ERG Variants

TMPRSS2-ERG translocation variants were amplified with Taq DNA polymerase, purified with a QIAquick PCR Purification Kit (Qiagen, Düsseldorf, Germany), and cloned into the pCR2.1-TOPO vector using a TOPO TA Invitrogen Cloning kit. The resulting plasmids were used for the transformation of Escherichia coli XL1-blue cells; positive clones were grown in LB medium overnight. The recombinant plasmids were isolated by the DM miniprep method [50 (link)] and sequenced in both directions using M13 forward and reverse primers (Invitrogen) and ABI BigDye Terminator Cycle Sequencing Kit v3.1 (Thermo Fisher, Waltham, MA, USA) with a Gene Amp 9700 PCR System (Thermo Fisher, Carlsbad, CA, USA). The sequences were detected with an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA USA). The sequences of TMPRSS2 (NM_005656) and ERG (NM_004449) were analyzed with the BLAST software (National Center for Biotechnology Information, NIH, GOV, USA).