Abstract
BackgroundPCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology.ResultsHere we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics.ConclusionsThe combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria.
Highlights
PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities
The detection, identification, and characterization of bacterial populations is an important goal in analytical microbiology
Other methods to assess the molecular composition of an environmental DNA sample, such as thermal or denaturing gradient gel electrophoresis (DGGE) [2], single stranded conformational polymorphism (SSCP) [3] heteroduplex analysis [4,5], or terminal restriction fragment (T-RFLP or TRF) analysis [6–9], are more rapid and amenable to large-scale experiments
Summary
PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and amenable to field-scale experiments. Other methods to assess the molecular composition of an environmental DNA sample, such as thermal or denaturing gradient gel electrophoresis (DGGE) [2], single stranded conformational polymorphism (SSCP) [3] heteroduplex analysis [4,5], or terminal restriction fragment (T-RFLP or TRF) analysis [6–9], are more rapid and amenable to large-scale experiments. The recent development of the DNA microarray technology has added a high throughput experimental format, potentially with great sensitivity [10–12]
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