Figure 2
Schematic representation of run through stop DNA mutagenesis and splicing technique with Ara-C. In this example two pieces of DNA are to be spliced (5' and 3' DNA segments) and mutated with an insertion of additional DNA. The 5' segment is amplified using PCR primers A (sense) and Ara-C2-A (anti-sense). Primer Ara-C2-A is designed to contain hybrid DNA with two adjacent molecules of Ara-C to stall polymerisation and produce a 5' overhang. Mutational addition is also incorporated into this primer. (Note that in this paper we created a mutational deletion in the human cofilin 1 gene, but here for illustration purpose, we describe a mutational addition). The 3' segment is amplified using PCR primers Ara-C2-B (sense) and B (anti-sense). Primer Ara-C2-B contains overlapping sequence with primer Ara-C2-A, and 2 molecules of Ara-C are incorporated to stall polymerization and produce a 5' overhang that is complementary to the overhang in Ara-C2-A. Both Ara-C primers are phosphorylated for down stream ligation. Since two adjacent Ara-C molecules produce moderate termination, PCR products contain a mixture of 5' overhang and blunt end DNA. Each PCR product is gel-isolated and subjected to short ligation, where cohesive end ligation is predominant. A portion of the ligation reaction is then subjected to a second PCR reaction, using primers A and B that span the entire mutated chimeric DNA. As mentioned above, 2 Ara-C molecules are moderate polymerization terminators. This assures that at the first round of the second PCR, the polymerase will run through the Ara-C in the template and incorporate native dGMP, that will ensure in turn proper polymerization in the next rounds and a product that will contain the native dCMP. For cloning purposes of the final PCR product, primers A abd B can include restriction sites (as used in this study). Alternatively, by using Taq polymerase in the second PCR reaction, the product can be cloned into TA cloning plasmids. Another alternative is to design primers A and B to contain at least 2 molecules of Ara-C to produce 5' overhangs to match cloning plasmids.