Selection for 3′ end triplets for polymerase chain reaction primers

The 3′ end of a primer is a key component of PCR primer design. Many recommendations for the composition and sequence of the 3′ end have been suggested based on theoretical considerations, but have not been verified experimentally. We analyzed 3′ end triplets of PCR primer sequences obtained from refereed journal articles, to test those recommendations and to make empirical recommendations for primer design. The frequencies of the 64 possible 3′end triplets among 2137 PCR primers from the VirOligo database were not uniformly distributed. From the analysis, we found that unfavored and preferred 3′ end triplets existed, and that the apparent preferences were not due to base compositions in viral genome sequences. Comparison of the sequences preferred by practitioners to those recommended, suggested that no single recommendation is entirely satisfactory. We suggest that recommendations be replaced with a scoring system incorporating empirical frequencies such as those reported here.