Bias in topoisomerase (TOPO)-cloning of multitemplate PCR products using locked nucleic acid (LNA)-substituted primers

► Effects of LNA substitution on topoisomerase cloning of amplicons are described. ► 3′ overhangs were missing in Taq-amplified PCR products. ► Topoisomerase-based cloning efficiency was affected in a sequence dependent manner. ► Topoisomerase cloning of LNA-substituted amplicons is prone to cloning bias.