PCR effects of melting temperature adjustment of individual primers in degenerate primer pools.

ABSTRACT: Deep sequencing of small subunit ribosomal RNA (SSU rRNA) gene amplicons continues to be the most common approach for characterization of complex microbial communities. PCR amplifications of conserved regions of SSU rRNA genes often employ degenerate pools of primers to enable targeting of a broad spectrum of organisms. One little noticed feature of such degenerate primer sets is the potential for a wide range of melting temperatures between the primer variants. The melting temperature variation of primers in a degenerate pool could lead to variable amplification efficiencies and PCR bias. Thus, we sought to adjust the melting temperature of each primer variant individually. Individual primer modifications were used to reduce theoretical melting temperature variation between primers, as we

SUBMITTER: Naqib A 

PROVIDER: S-EPMC6404654 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature