The length limit of 5' nucleotide additions to PCR primers

The addition of nucleotides to polymerase chain reaction (PCR) primers has become a widely used technique to facilitate the cloning of PCR products. A long fragment mismatched with template, such as an epitope-encoding sequence, needs to be added to the 5′ end of the primer for identification and purification of the expressed protein. However, the length limit of added sequences has not yet been determined. Under our condition, the maximum length for 5′ nucleotide additions was found to be 108, when a fully matched forwards primer and partially matched reverse primer contained 44.4–55.6% GC in the matched region with template are used. The results shown here are very useful for scientists who want to add a long mismatched sequence with template, such as an epitop/a signal peptide encoding sequence and/or the multiple cloning sites.