© 2012 Landes Bioscience. Do not distribute. Figure 7. Primer extension using templates T4-T7. Lanes 1, 3, 5 and 7: positive controls (dATP, dGTP, dCTP and TTP); lanes 2, 4, 6 and 8: negative controls [dGTP, dCTP and dTTP (for incorporation across a-L-LNA-T); or dATP, dCTP and dTTP (for incorporation across a-L-LNA-5-methyl-C)]; lanes 1 and 2: template T4; lanes 3 and 4: template T5; lanes 5 and 6: template T6; lanes 7 and 8: template T7; lane 9: P1 and T1 (19mer and 43mer). modified template strand. In general, primer extension was not halted at the position of the a-L-LNA nucleotide but rather at the subsequent position. Perhaps the high stability of the a-L-LNA: DNA pairing and/or the unique structure of the a-L-LNA nucleotides prevent the flexibility needed to position the primer strand 3'-oxygen correctly in the active site of the polymerases for further extension. Comparison with LNA nucleotide incorporation. Of the seven polymerases tested for their ability to incorporate a-L-LNA nucleo- tides, the three B-family polymerases (KOD, 9°Nm and Phusion) proved to be by far the most efficient. This is not to say that other B-family polymerases will be efficient too as Deep Vent and Therminator proved to be poor at incorporating LNA nucleotides, contrary to KOD, 9°Nm and Phusion DNA polymerases.18 18 Artificial DNA: PNA & XNA Volume 3 Issue 1