Figure 2

PCR-ligation strategy for generation of TNC-16 and TNC-14/16 clones. (a) Primers designed incorporating unique restriction sites were used to link exons 9 to 16 in a three-step polymerase chain reaction (PCR)-mediated ligation strategy. This allowed directional cloning from the Bcl1 and Sfi1 sites into the TNC-S sequence and this sequence was subsequently transferred into the mammalian expression vector pCMV script. The gel image shows exon 9 and exon 16 products and the combined 9–16 amplicon. (b) The same PCR-mediated ligation strategy was used to link exons 9, 14 and 16 prior to directional cloning into the TNC-S sequence for expression. The gel image shows the multi-step process used to link exons 9, 14 and 16.