Optimized Design of Degenerate Primers for PCR Based on DNA or Protein Sequence Comparisons

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in three different applications, including the use of specific determinants at the 3'-end, to (i) improve PCR efficiency with related sequences for members of a protein family by complete degeneration at a core box of conserved genetic information at the 3'-end with the reduction of degeneration at the 5'-end, (ii) optimize specificity of allelic discrimination of closely related DNA sequences of orthologous by 5'-end fully degenerate primers, and (iii) increase the PCR efficiency of primers by targeting DNA sequences belonging to specific phylogenetic groups, within a large and diverse gene family, allowing the use of multiplex/degenerate PCR.