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: 2.12). 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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    DESCRIPTION: Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes
    Full-text · Research · Dec 2015
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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. "

    Full-text · Dataset · Aug 2015
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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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    ABSTRACT: Background: Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. Results: A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen nsupported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0–20%), showing maximal growth and enzyme production at 0–5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). Conclusions: A newhalotolerant alkaliphilic alkaline protease producing Bacillus sp.NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications. © 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved
    Full-text · Article · Apr 2015 · Electronic Journal of Biotechnology
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