Deconstruction of Polymerase Chain Reaction to Reduce Bias Associated with Degenerate Primers

The polymerase chain reaction(PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA templates. To target the high taxonomic and genetic diversity in microbial communities, degenerate primers are frequently used. The use of such primers can lead to inefficient PCR amplification of genes from some taxa, resulting in a distortion of the true relative abundance of target genes. In this thesis, a new mechanism for PCR amplification of genes using degenerate primers was developed and tested to determine if amplification biases could be reduced. PCR yields were analyzed using next-generation sequencing to determine PCR efficiency. Sequence data generated from mock community DNA and unknown environmental genomic DNA were analyzed using custom bioinformatics pipelines to determine the effect of PCR strategy on relative abundance of known and unknown taxa in samples. We demonstrate here that the new two-stage PCR amplification strategy developed here greatly reduces the effect of primer biases in amplification of mock DNA communities of known concentration and abundance, relative to standard amplification reactions. In addition, we show that this method is also able to increase amplification of templates with 3’ mismatches relative to all primers, thereby increasing the potential for primers to target a broader range of taxa. The new method was employed to analyze microbial communities from unknown environmental genomic DNA samples and our analyses demonstrated that the method is effective for analysis of complex microbial communities. The advantages of this strategy touch many areas of research- The method is simple to perform and is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup. We also establish that there is better capture including PCR with degenerate primers, PCR with primers potentially containing mismatches (including single nucleotide polymorphisms,SNPs) to known and unknown templates, multiplex PCR for target capture, and quantitative PCR with degenerate primers.