title: The Use of Degenerate Primers in qPCR Analysis of Functional Genes Can Cause Dramatic Quantification Bias as Revealed by Investigation of nifH Primer Performance.
abstract: The measurement of functional gene abundance in diverse microbial communities often employs quantitative PCR (qPCR) with highly degenerate oligonucleotide primers. While degenerate PCR primers have been demonstrated to cause template-specific bias in PCR applications, the effect of such bias on qPCR has been less well explored. We used a set of diverse, full-length nifH gene standards to test the performance of several universal nifH primer sets in qPCR. We found significant template-specific bias in all but the PolF/PolR primer set. Template-specific bias caused more than 1000-fold mis-estimation of nifH gene copy number for three of the primer sets and one primer set resulted in more than 10,000-fold mis-estimation. Furthermore, such template-specific bias will cause qPCR estimates to vary in response to beta-diversity, thereby causing mis-estimation of changes in gene copy number. A reduction in bias was achieved by increasing the primer concentration. We conclude that degenerate primers should be evaluated across a range of templates, annealing temperatures, and primer concentrations to evaluate the potential for template-specific bias prior to their use in qPCR.
authors.affiliation: School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA.
authors.forename: John Christian
authors.initials: JC
authors.lastname: Gaby
authors.affiliation: School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA. dhb28@cornell.edu.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Buckley
mesh_terms: D001419:Bacteria / Q000145:classification*; D001426:Bacterial Proteins / Q000032:analysis*; D017931:DNA Primers / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D008828:Microbiological Techniques / Q000379:methods; D010088:Oxidoreductases / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D010088:Oxidoreductases; C029130:nitrogenase reductase
keywords: Amplification bias; Degenerate primers; nifH; qPCR