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

PeerJ

article

Ankur Naqib¹  Trisha Jeon¹  Kevin Kunstman¹  Weihua Wang¹  Yiding Shen¹  Dagmar Sweeney¹  Marieta Hyde¹  Stefan J. Green¹ 

[1] Sequencing Core, Research Resources Center, University of Illinois at Chicago

关键词: Microbiome;    phiX;    Degenerate primers;    NGS;    Primer design;    16S rRNA;   

DOI  :  10.7717/peerj.6570

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

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 well as to introduce inter-cluster nucleotide diversity during Illumina sequencing of primer regions. We demonstrate here the suitability of such primers for microbial community analysis. However, no substantial differences in microbial community structure were revealed when using primers with adjusted melting temperatures, though the optimal annealing temperature decreased.

【 授权许可】

CC BY