GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects

The amplification of GC-rich templates by any PCR method is usually a difficult task and despite the development of modified methods and conditions, this type of amplification still remains a specific case approach. Problems usually observed with GC-rich DNA are constraint of template amplification by stable secondary structures that stall or reduce the DNA polymerase progress, and the presence of secondary annealing sites giving rise to nonspecific amplified bands. This latter point is not exclusive to GC-rich templates but is frequently encountered in other types of templates. In order to design a more general method for GC-rich templates, different DNA polymerases were compared in combination with different organic solvents with the purpose of abolishing stable secondary structures (1 ). Our attention focused on the inverse polymerase chain reaction (iPCR) used to perform site-directed mutagenesis (1 2 ). This very attractive method req⪚res a single pair of primers and involves the amplification of the whole recombinant plasmid, a difficult step with high GC-content DNA. Inverse PCR also proves useful in cloning missing parts of genes by using a self-ligated genomic DNA fragment as template.