Article

PCR-generated artefact from 16S rRNA gene-specific primers. FEMS Microbiol Lett

Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia.
FEMS Microbiology Letters (Impact Factor: 2.72). 08/2005; 248(2):183-7. DOI: 10.1016/j.femsle.2005.05.043
Source: PubMed

ABSTRACT Artefacts consisting of concatenated oligonucleotide primer sequences were generated during sub-optimally performing polymerase chain reaction amplification of bacterial 16S rRNA genes using a commonly employed primer pair. These artefacts were observed during amplification for terminal restriction fragment length polymorphism analyses of complex microbial communities, and after amplification from DNA from a microbial culture. Similar repetitive motifs were found in gene sequences deposited in GenBank. The artefact can be avoided by using different primers for the amplification reaction.

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    • "Total DNA was extracted using DNeasy Blood & Tissue Kits (Qiagen, USA) according to the manufacturer's instructions. Eubacterial-specific forward primer: 16F27 (5′-AGA GTT TGA TCC TGG CTC AG-3′), and reverse primer: 16R1525 (5′-AAG GAG GTG ATC CAG CCG CA-3′) were used to amplify 16S rDNA gene [20] [21]. PCR amplification was performed in a final reaction volume of 50 μL. "
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    • "Total DNA was extracted using DNeasy Blood & Tissue Kits (Qiagen, USA) according to the manufacturer's instructions. Eubacterial-specific forward primer: 16F27 (5′-AGA GTT TGA TCC TGG CTC AG-3′), and reverse primer: 16R1525 (5′-AAG GAG GTG ATC CAG CCG CA-3′) were used to amplify 16S rDNA gene [20] [21]. PCR amplification was performed in a final reaction volume of 50 μL. "
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