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Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

Oldham AL, Drilling HS, Stamps BW, Stevenson BS, Duncan KE - AMB Express (2012)

Bottom Line: The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition.Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach.Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Microbiology and Plant Biology, University of Oklahoma, 770 Van Vleet Oval GLCH #136, Norman, OK 73019, USA. athenia.L.oldham-1@ou.edu.

ABSTRACT
The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

No MeSH data available.


Agarose gel analysis of sample A DNA extracts. Comparison of DNA fragment size and relative quantity among subsamples. An aliquot (10 μl) of DNA extracted from replicate subsamples of sample A using the a) PowerBiofilm, b) QuickGene and c) Maxwell methods. Low weight nucleic acid (RNA) species in Maxwell extractions are indicated by an asterisk. Sizes (kb) of bands in the Lambda DNA/EcoRI+HindIII marker (left lane) are indicated.
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Figure 1: Agarose gel analysis of sample A DNA extracts. Comparison of DNA fragment size and relative quantity among subsamples. An aliquot (10 μl) of DNA extracted from replicate subsamples of sample A using the a) PowerBiofilm, b) QuickGene and c) Maxwell methods. Low weight nucleic acid (RNA) species in Maxwell extractions are indicated by an asterisk. Sizes (kb) of bands in the Lambda DNA/EcoRI+HindIII marker (left lane) are indicated.

Mentions: Biofilm material scraped from the inner surface of three separate oil pipelines were initially extracted as ten subsamples to compare extraction reproducibility among replicate samples. The DNA fragment was visualized by gel electrophoresis and the gel images for sample A extracts are shown for a visual comparison between the three platforms (Figure 1). The PowerBiofilm method (P) extracted appreciable amounts of DNA from the ten subsamples, but DNA yields varied widely among them (Figure 1a). This result indicated a low consistency in extraction among replicate samples. By contrast, the QuickGene platform demonstrated more uniformity among subsamples (Figure 1, compare panel a to b), as did the Maxwell (Figure 1, compare panel a to c). RNA was also extracted using the Maxwell system, and was visible as a low molecular weight band (Figure 1c).


Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

Oldham AL, Drilling HS, Stamps BW, Stevenson BS, Duncan KE - AMB Express (2012)

Agarose gel analysis of sample A DNA extracts. Comparison of DNA fragment size and relative quantity among subsamples. An aliquot (10 μl) of DNA extracted from replicate subsamples of sample A using the a) PowerBiofilm, b) QuickGene and c) Maxwell methods. Low weight nucleic acid (RNA) species in Maxwell extractions are indicated by an asterisk. Sizes (kb) of bands in the Lambda DNA/EcoRI+HindIII marker (left lane) are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539857&req=5

Figure 1: Agarose gel analysis of sample A DNA extracts. Comparison of DNA fragment size and relative quantity among subsamples. An aliquot (10 μl) of DNA extracted from replicate subsamples of sample A using the a) PowerBiofilm, b) QuickGene and c) Maxwell methods. Low weight nucleic acid (RNA) species in Maxwell extractions are indicated by an asterisk. Sizes (kb) of bands in the Lambda DNA/EcoRI+HindIII marker (left lane) are indicated.
Mentions: Biofilm material scraped from the inner surface of three separate oil pipelines were initially extracted as ten subsamples to compare extraction reproducibility among replicate samples. The DNA fragment was visualized by gel electrophoresis and the gel images for sample A extracts are shown for a visual comparison between the three platforms (Figure 1). The PowerBiofilm method (P) extracted appreciable amounts of DNA from the ten subsamples, but DNA yields varied widely among them (Figure 1a). This result indicated a low consistency in extraction among replicate samples. By contrast, the QuickGene platform demonstrated more uniformity among subsamples (Figure 1, compare panel a to b), as did the Maxwell (Figure 1, compare panel a to c). RNA was also extracted using the Maxwell system, and was visible as a low molecular weight band (Figure 1c).

Bottom Line: The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition.Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach.Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Microbiology and Plant Biology, University of Oklahoma, 770 Van Vleet Oval GLCH #136, Norman, OK 73019, USA. athenia.L.oldham-1@ou.edu.

ABSTRACT
The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

No MeSH data available.