Fingerprinting of prokaryotic 16S rRNA genes using oligodeoxyribonucleotide microarrays and virtual hybridization

Miguel Ángel Reyes-López, Alfonso Méndez-Tenorio, Rogelio Maldonado-Rodríguez, Mitchel J. Doktycz, James T. Fleming, Kenneth L. Beattie

Research output: Contribution to journalReview articlepeer-review

29 Scopus citations

Abstract

An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5′ end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single-stranded, fluorescence-tagged PCR products were hybridized to the microarrays at 15°C. The experimental results were compared with the ΔG° values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a 'virtual hybridization' software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated ΔG° values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.

Original languageEnglish
Pages (from-to)779-789
Number of pages11
JournalNucleic Acids Research
Volume31
Issue number2
DOIs
StatePublished - Jan 15 2003

Funding

The authors thank Brent Harker of ORNL for arraying the probes onto glass slides, and thank Aaron Nagel of The University of Tennessee for providing valuable technical advice. The authors thank Engineer Armando Guerra-Trejo of the ENCB-IPN for substantial assistance with statistical analysis. Financial support for this work was provided by CONACYT and IPN at ENCB-IPN and by the US Department of Defense Strategic Environmental Research and Development Program (CU-1081) at ORNL.

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