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A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities.

Harrington CR, Lucchini S, Ridgway KP, Wegmann U, Eaton TJ, Hinton JC, Gasson MJ, Narbad A - BMC Microbiol. (2008)

Bottom Line: We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides.Short oligonucleotides consistently gave higher specificity.An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Commensals and Microflora, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, Norfolk, NR4 7UA, UK. Carl.Harrington@bbsrc.ac.uk

ABSTRACT

Background: The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.

Results: We have developed a culture-independent, semi-quantitative, rapid method for detection of gut bacterial populations based on 16S rDNA probes using a DNA microarray. We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides. Short oligonucleotides consistently gave higher specificity. Optimal DNA amplification and labelling, hybridisation and washing conditions were determined using a probe with an increasing number of nucleotide mismatches, identifying the minimum number of nucleotides needed to distinguish between perfect and mismatch probes. An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population. The sensitivity of the microarray was determined to be 8.8 x 104 bacterial cells g-1 faecal sample, which is more sensitive than a number of existing profiling methods. The short oligonucleotide microarray was used to compare the faecal flora from healthy individuals and a patient suffering from Ulcerative Colitis (UC) during the active and remission states. Differences were identified in the bacterial profiles between healthy individuals and a UC patient. These variations were verified by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing.

Conclusion: In this study we demonstrate the design, testing and application of a highly sensitive, short oligonucleotide community microarray. Our approach allows the rapid discrimination of bacteria inhabiting the human GI tract, at taxonomic levels ranging from species to the superkingdom bacteria. The optimised protocol is available at: http://www.ifr.ac.uk/safety/microarrays/#protocols. It offers a high throughput method for studying the dynamics of the bacterial population over time and between individuals.

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Related in: MedlinePlus

Determination of the optimum washing temperature for the short oligonucleotide probes. Discrimination of the perfect match probe (BLON135a), against either the 1 nt (BLON135b) (grey bars) or 2 nt (BLON135c) (white bars) mismatch probes. Labelled 16S ribosomal DNA from a pure culture of Bifidobacterium longum was hybridised at 58°C and washed between 55°C and 66°C. Perfect match probe intensity at each different temperature was then divided either by the 1 nt or 2 nt corresponding mismatch binding, and error bars represent the standard error.
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Figure 2: Determination of the optimum washing temperature for the short oligonucleotide probes. Discrimination of the perfect match probe (BLON135a), against either the 1 nt (BLON135b) (grey bars) or 2 nt (BLON135c) (white bars) mismatch probes. Labelled 16S ribosomal DNA from a pure culture of Bifidobacterium longum was hybridised at 58°C and washed between 55°C and 66°C. Perfect match probe intensity at each different temperature was then divided either by the 1 nt or 2 nt corresponding mismatch binding, and error bars represent the standard error.

Mentions: In order to confirm the optimal hybridisation and washing conditions for the short probes, we performed DNA melting curves. The labelled 16S ribosomal gene from a pure culture of Bifidobacterium longum was hybridised to the corresponding probes containing 0, 1 or 2 nt mismatches. This established that hybridisation at 58°C gave the best discrimination (unpublished data). A range of washing temperatures were examined and again confirmed that 58°C was most selective for hybridisation (Figure 1 and Figure 2). As with the pure culture specificity testing, hybridisations and washes at higher temperatures resulted in greater specificity, but the intensity of the specific signal was reduced leading to increased noise.


A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities.

Harrington CR, Lucchini S, Ridgway KP, Wegmann U, Eaton TJ, Hinton JC, Gasson MJ, Narbad A - BMC Microbiol. (2008)

Determination of the optimum washing temperature for the short oligonucleotide probes. Discrimination of the perfect match probe (BLON135a), against either the 1 nt (BLON135b) (grey bars) or 2 nt (BLON135c) (white bars) mismatch probes. Labelled 16S ribosomal DNA from a pure culture of Bifidobacterium longum was hybridised at 58°C and washed between 55°C and 66°C. Perfect match probe intensity at each different temperature was then divided either by the 1 nt or 2 nt corresponding mismatch binding, and error bars represent the standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2628385&req=5

Figure 2: Determination of the optimum washing temperature for the short oligonucleotide probes. Discrimination of the perfect match probe (BLON135a), against either the 1 nt (BLON135b) (grey bars) or 2 nt (BLON135c) (white bars) mismatch probes. Labelled 16S ribosomal DNA from a pure culture of Bifidobacterium longum was hybridised at 58°C and washed between 55°C and 66°C. Perfect match probe intensity at each different temperature was then divided either by the 1 nt or 2 nt corresponding mismatch binding, and error bars represent the standard error.
Mentions: In order to confirm the optimal hybridisation and washing conditions for the short probes, we performed DNA melting curves. The labelled 16S ribosomal gene from a pure culture of Bifidobacterium longum was hybridised to the corresponding probes containing 0, 1 or 2 nt mismatches. This established that hybridisation at 58°C gave the best discrimination (unpublished data). A range of washing temperatures were examined and again confirmed that 58°C was most selective for hybridisation (Figure 1 and Figure 2). As with the pure culture specificity testing, hybridisations and washes at higher temperatures resulted in greater specificity, but the intensity of the specific signal was reduced leading to increased noise.

Bottom Line: We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides.Short oligonucleotides consistently gave higher specificity.An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population.

View Article: PubMed Central - HTML - PubMed

Affiliation: Commensals and Microflora, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, Norfolk, NR4 7UA, UK. Carl.Harrington@bbsrc.ac.uk

ABSTRACT

Background: The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.

Results: We have developed a culture-independent, semi-quantitative, rapid method for detection of gut bacterial populations based on 16S rDNA probes using a DNA microarray. We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides. Short oligonucleotides consistently gave higher specificity. Optimal DNA amplification and labelling, hybridisation and washing conditions were determined using a probe with an increasing number of nucleotide mismatches, identifying the minimum number of nucleotides needed to distinguish between perfect and mismatch probes. An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population. The sensitivity of the microarray was determined to be 8.8 x 104 bacterial cells g-1 faecal sample, which is more sensitive than a number of existing profiling methods. The short oligonucleotide microarray was used to compare the faecal flora from healthy individuals and a patient suffering from Ulcerative Colitis (UC) during the active and remission states. Differences were identified in the bacterial profiles between healthy individuals and a UC patient. These variations were verified by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing.

Conclusion: In this study we demonstrate the design, testing and application of a highly sensitive, short oligonucleotide community microarray. Our approach allows the rapid discrimination of bacteria inhabiting the human GI tract, at taxonomic levels ranging from species to the superkingdom bacteria. The optimised protocol is available at: http://www.ifr.ac.uk/safety/microarrays/#protocols. It offers a high throughput method for studying the dynamics of the bacterial population over time and between individuals.

Show MeSH
Related in: MedlinePlus