Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: A tool for rapid bacterial identification

Abstract

A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNAglycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-offlight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yetuncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria. E merging antibiotic-resistant pathogens, the actual threat of bioterrorist attacks, and bioremediation or bioprospecting efforts all require fast and accurate identification of the bacteria involved. Conventional diagnoses rely on the characterization of phenotypic traits of pure cultures obtained from specimens after cultivation and isolation of bacteria on appropriate laboratory media. These culture-based methods are time-consuming especially for slow-growing pathogens such as mycobacteria and generally are restricted to yet-culturable bacteria, which constitute only a small fraction (1-10%) of the global bacterial diversity (1). In contrast, genotypic analyses such as comparative sequencing of PCR-amplified 16S rRNA genes (rDNAs) allow for the identification of both cultured and as-yet-uncultured bacteria (1). Currently, 16S rDNA sequences constitute the largest gene-specific data set, and the number of entries in generally accessible databases is increasing continually (currently Ϸ30,000), making 16S rDNA-based identification of unknown bacteria isolates more and more likely. Conventional 16S rDNA sequencing is expensive and time-consuming because of the enzymatic reactions and electrophoresis or chromatography steps involved. Hence, it still is not suited for massive parallel testing as required in clinical, pharmaceutical, or environmental microbiology. Compared with standard 16S rDNA sequencing, alternative nonelectrophoretic techniques such as sequencing by MS (2) or pyrosequencing (3) are faster but generate only short read lengths, limiting their utility in 16S rDNA-based bacterial identification.Here we introduce the concept of base-specific fragmentation of PCR-amplified DNA follow...

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

“…The 6-aza-2-thiothymine (6-ATT), 2',4',6'-trihydroxyacetophnone (2,4,6-THAP), 2',3',4'-trihydroxyacetophnone (2,3,4-THAP), and ammonium citrate were also obtained from Aldrich Chemical Co. and used without further purification. To minimize production of metal-ion adducts, a few beads of cation-exchange resin in the NH 4 ϩ form were usually added to the oligonucleotide analyte prior to the mass spectrometric analysis. They were prepared from chromatography beads (AG50W-X8, 100 -200 mesh; Bio-Ad, Melville, NY) in the H ϩ form by using a literature procedure [20].…”

“…Two recent reports show that the use of enzymes to release small oligodeoxynucleotides followed by simple mass measurement [2,3] can be an effective approach to locating abasic sites. Others used enzymes to produce abasic sites and made use of the ease of alkaline cleavage at those sites to produce small ODNs, which can be mass-measured by matrix-assisted laser desorption ionization (MALDI) mass spectrometry [4]. Another recent development of a physical method for detecting abasic sites on DNA is atomic force microscopy [5].…”

Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing

“…MALDI-TOF MS sequence-based typing for high-level discrimination of individual microbial taxa based on signatures within variable regions in the 16S rDNA gene region has previously been applied to discriminate mycobacteria and Bordetella species (12,13).…”

Typing of single nucleotide polymorphisms by MALDI mass spectrometry: Principles and diagnostic applications

“…A similar approach was applied to bacterial identification using the 16S rRNA gene (16S rDNA) [58]. Base-specific fragmentation of PCR products using uracil-DNA-glycosylase was combined with MALDI detection.…”