Limits...
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.


Illustration of DNA extraction variability among replicate subsamples. Box and whisker plots of replicate extractions (n = 10) for a) sample A, b) sample B, and c) sample C using PowerBiofilm (P), QuickGene (Q) and Maxwell (M) methods. The lower and upper whiskers illustrate minimum and maximum yields, respectively and the median yield separates the box into upper and lower quartiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3539857&req=5

Figure 2: Illustration of DNA extraction variability among replicate subsamples. Box and whisker plots of replicate extractions (n = 10) for a) sample A, b) sample B, and c) sample C using PowerBiofilm (P), QuickGene (Q) and Maxwell (M) methods. The lower and upper whiskers illustrate minimum and maximum yields, respectively and the median yield separates the box into upper and lower quartiles.

Mentions: DNA yields were estimated for each extraction using Qubit fluorometry and box-plots were used to illustrate the level of variability in DNA yields among the ten replicate DNA extracts (Figure 2). For sample A PowerBiofilm extracts, DNA yields ranged more than 10-fold from 0.06 μg to 1.17 μg, with the median yield at 0.17 μg (Figure 2a). For QuickGene and Maxwell extracts, median values were higher than PowerBiofilm and DNA yields were more consistent among the replicates. For samples B and C, distances between upper and lower whiskers (i.e. maximum and minimum yields, respectively) were closest among replicates for the Maxwell and PowerBiofilm extracts, respectively (Figures 2b and 2c). Subsequently, the ten replicate DNA extracts from each platform were pooled to compare the total DNA yields from equivalent starting sample volumes. The total amount of DNA extracted from 5 ml of sample A was approximately 3.37 μg using PowerBiofilm. Yields were higher for QuickGene and Maxwell at 8.01 μg and 6.01 μg, respectively. For sample B, the automated platforms also increased DNA yields from 0.94 μg (PowerBiofilm) to 12.56 μg and 5.80 μg for Maxwell and QuickGene, respectively. Next, DNA yields from the lower biomass sample C extractions were compared. From 10 ml of sample C, DNA yields were comparable for PowerBiofilm and QuickGene at 100 ng and 130 ng, respectively, however DNA yield was increased by almost ten-fold (870 ng) using the Maxwell platform. Together, these data demonstrated that the Maxwell platform could increase the DNA yields from both the high- and lower-biomass samples. QuickGene could also increase yields from samples A and B but had a negligible effect on the low-biomass sample C. Together, these results demonstrated that more automated platforms extracted higher DNA yields than the manual approach from equivalent starting sample volumes.


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)

Illustration of DNA extraction variability among replicate subsamples. Box and whisker plots of replicate extractions (n = 10) for a) sample A, b) sample B, and c) sample C using PowerBiofilm (P), QuickGene (Q) and Maxwell (M) methods. The lower and upper whiskers illustrate minimum and maximum yields, respectively and the median yield separates the box into upper and lower quartiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Illustration of DNA extraction variability among replicate subsamples. Box and whisker plots of replicate extractions (n = 10) for a) sample A, b) sample B, and c) sample C using PowerBiofilm (P), QuickGene (Q) and Maxwell (M) methods. The lower and upper whiskers illustrate minimum and maximum yields, respectively and the median yield separates the box into upper and lower quartiles.
Mentions: DNA yields were estimated for each extraction using Qubit fluorometry and box-plots were used to illustrate the level of variability in DNA yields among the ten replicate DNA extracts (Figure 2). For sample A PowerBiofilm extracts, DNA yields ranged more than 10-fold from 0.06 μg to 1.17 μg, with the median yield at 0.17 μg (Figure 2a). For QuickGene and Maxwell extracts, median values were higher than PowerBiofilm and DNA yields were more consistent among the replicates. For samples B and C, distances between upper and lower whiskers (i.e. maximum and minimum yields, respectively) were closest among replicates for the Maxwell and PowerBiofilm extracts, respectively (Figures 2b and 2c). Subsequently, the ten replicate DNA extracts from each platform were pooled to compare the total DNA yields from equivalent starting sample volumes. The total amount of DNA extracted from 5 ml of sample A was approximately 3.37 μg using PowerBiofilm. Yields were higher for QuickGene and Maxwell at 8.01 μg and 6.01 μg, respectively. For sample B, the automated platforms also increased DNA yields from 0.94 μg (PowerBiofilm) to 12.56 μg and 5.80 μg for Maxwell and QuickGene, respectively. Next, DNA yields from the lower biomass sample C extractions were compared. From 10 ml of sample C, DNA yields were comparable for PowerBiofilm and QuickGene at 100 ng and 130 ng, respectively, however DNA yield was increased by almost ten-fold (870 ng) using the Maxwell platform. Together, these data demonstrated that the Maxwell platform could increase the DNA yields from both the high- and lower-biomass samples. QuickGene could also increase yields from samples A and B but had a negligible effect on the low-biomass sample C. Together, these results demonstrated that more automated platforms extracted higher DNA yields than the manual approach from equivalent starting sample volumes.

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.