Thermal-bias PCR: generation of amplicon libraries without degenerate primer interference

The polymerase chain reaction (PCR) has been used to amplify specific gene regions for many taxonomic studies and there have been substantial efforts to develop protocols that efficiently amplify target regions from a majority of mixed-template populations. Most protocols include the use of degenerate oligonucleotide primer pools, which contain mixed nucleotide sequences to improve priming from templates containing non-consensus sequence variations in their primer-binding sites. In this work, computational modeling and experimental measurements revealed that degenerate primers reduce efficiency well before a substantial product pool has been generated. It was also discovered that non-degenerate primers produced amplicons significantly better than their degenerate counterparts when amplifying either a consensus or a non-consensus target. Using quantitative, real-time PCR (qPCR) and data fitting as a guide, a new PCR protocol was developed that avoids the use of degenerate primers and allows for the stable amplification of targets containing mismatches to the targeting primers. This protocol involves the use of only two non-degenerate primers with no intermediate processing steps and it allows for the reproducible production of amplicon sequencing libraries that maintain the fractional representations of rare members.