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New primers for the class Actinobacteria: Application to marine and terrestrial environments

Lookup NU author(s): Dr James Stach, Dr Luis Maldonado, Emeritus Professor Alan Ward, Professor Michael Goodfellow, Professor Alan Bull

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Abstract

In this study, we redesigned and evaluated primers for the class Actinobacteria. In silico testing showed that the primers had a perfect match with 82% of genera in the class Actinobacteria, representing a 26-213% improvement over previously reported primers. Only 4% of genera that displayed mismatches did so in the terminal three bases of the 3′ end, which is most critical for polymerase chain reaction success. The primers, designated S-C-Act-0235-a-S-20 and S-C-Act-0878-a-A-19, amplified an ≈640 bp stretch of the 16S rRNA gene from all actinobacteria tested (except Rubrobacter radiotolerans) up to an annealing temperature of 72°C. An Actinobacteria Amplification Resource (http://microbe2.ncl.ac.uk/MMB/AAR.htm) was generated to provide a visual guide to aid the amplification of actinobacterial 16S rDNA. Application of the primers to DNA extracted from marine and terrestrial samples revealed the presence of actinobacteria that have not been described previously. The use of 16S rDNA similarity and DNA-DNA pairing correlations showed that almost every actinomycete clone represented either a new species or a novel genus. The results of this study reinforce the proposition that current culture-based techniques drastically underestimate the diversity of Actinobacteria in the environment and highlight the need to evaluate taxon-specific primers regularly in line with improvements in databases holding 16S rDNA sequences.


Publication metadata

Author(s): Stach JEM, Maldonado LA, Ward AC, Goodfellow M, Bull AT

Publication type: Article

Publication status: Published

Journal: Environmental Microbiology

Year: 2003

Volume: 5

Issue: 10

Pages: 828-841

ISSN (print): 1462-2912

ISSN (electronic): 1462-2920

Publisher: Wiley-Blackwell

URL: http://dx.doi.org/10.1046/j.1462-2920.2003.00483.x

DOI: 10.1046/j.1462-2920.2003.00483.x

PubMed id: 14510836


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