Band smearing of PCR amplified bacterial 16S rRNA genes: dependence on initial PCR target diversity

Abstract

Band smearing in agarose gels of PCR amplified bacterial 16S rRNA genes is understood to comprise amplicons of varying sizes arising from PCR errors, and requires elimination. We consider that with amplified heterogeneous DNA, delayed electro-migration is caused not by PCR errors but by dsDNA structures that arise from imperfect strand pairing. The extent of band smearing was found to be proportional to the sequence heterogeneity in 16S rRNA variable regions. Denaturing alkaline gels showed that all amplified DNA was of the correct size. A novel bioinformatic approach was used to reveal that band smearing occurred due to imperfectly paired strands of the amplified DNA. Since the smear is a structural fraction of the correct size PCR product, it carries important information on richness and diversity of the target DNA. For accurate analysis, the origin of the smear must first be identified before it is eliminated by examining the amplified DNA in denaturing alkaline gels.

Keywords: 16S ribosomal RNA; Bacterial diversity; Bioinformatics; F1; F1-score; Gel electrophoresis; MCC; Matthews correlation coefficient; Metagenomics; NN; NOESY NMR; PCR; S(obs); SELEX; WC; average of the Watson–Crick NN free energy parameters; gap; gap-opening penalty; nearest neighbour (model); nuclear Overhauser effect spectroscopy nuclear magnetic resonance; observed number of OTUs; systematic evolution of ligands by exponential enrichment.