Scaling Up the Ligase Chain Reaction-Based Approach to Gene Synthesis

as a template for PCR. Sequencing of the plasmid DNA can now be performed using two PCR primers flanking the MCS of the vector (we used M13 universal forward primer and M13 universal reverse primer; Figure 1). If the unique primer is set 100–150 bp from the end of the known sequence, sufficient overlapping sequence is generated to align the sequences unambiguously. This method was used in our laboratory for several cycles of sequencing. In the overlapping sequence, all bases were identical, suggesting that this method is not only fast and inexpensive but also accurate.