Analysis of Double‐stranded Polymerase Chain Reaction Products from the Bacillus cereus Group by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Abstract

The analysis of polymerase chain reaction (PCR) products by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) has been achieved. Specifically, a 105 base-pair nucleotide portion of the ribosomal spacer region was amplified in two members of the B. cereus group (i.e. B. thuringiensis and B. cereus) using PCR. These amplified regions were then analyzed by gel electrophoresis and ESI-FTICR. Based on the predicted sequence of the PCR products for each organism, the mass measurement using ESI-FTICR matched the theoretical mass within experimental error and was consistent with gel electrophoresis results. In contrast, for the typical several hour time-scale of the gel electrophoresis experiment, the mass spectrometric analysis was completed in a matter of minutes. To our knowledge, this constitutes the first report demonstrating the ionization and detection of a double-stranded PCR product by ESI-MS. This preliminary result indicates the potential use of ESI-MS to analyze PCR products on a rapid time-scale, with potential for medical and taxonomic applications.

Nonlinearity in genetic decoding: Homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting

“…The nucleotide composition of the poly(A) region was accurately determined by measuring the molecular weight of PCR products using electrospray mass spectrometry (22)(23)(24). The electrospray ionization process generates a series of multiply-charged molecular ions from which very accurate (better than 0.01%) mass assignments are derived for each DNA molecule in a PCR experiment.…”

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry

“…The results of this exploratory investigation show that MALDI MS provides interesting possibilities for recombinant DNA research. The first report on combined PCR and MS of bacterial DNA concerned the ES MS analysis of double-stranded 105 base pair PCR products obtained from the ribosomal spacer region in B. thuringiensis and B. cereus [95]. In a wider study, by the same group, ES MS was used to determine the mass of the PCR amplification product of a specific region of the genome of various Bacillus spp.…”

“…Desalting was found crucial for successful use of ES MS, and further improvements in mass spectral quality were obtained by applying solvent additives in the spray. The FT-ICR mass spectrometer allowed accurate mass measurements of 105 base pair ds-DNA, for the distinction of B. thuringiensis from B. cereus [95], of 89 and 114 base pair ds-DNA, for the distinction of B. subtilis from B. thuringiensis, B. anthracis and B. cereus (PCR products from the latter three species should be identical; [96]) and of unexpectedly variable PCR products from B. cereus [97]. With the solvent compositions used, the ds-DNA PCR products appear in the spectrum as two charge state envelopes from two (complementary) single DNA strands; a typical example is given in Figure 9.…”

Monitoring Genetic and Metabolic Potential for In-Site Bioremediation: Mass Spectrometry

“…In the 1980's, advances in ionization processes, such as matrix-assisted laser desorption/ionization (MALDI) [Hillenkamp et al, 1991] and electrospray (ES) ionization [Fenn et al, 1989] allowed analysis of large biomolecules, including DNA oligomers, by mass spectrometry. When used for detection of PCR products, mass spectrometry yields information similar to electrophoresis, with DNA size information read directly from the peaks in the mass spectrum, but with analysis times reduced by several orders of magnitude compared to electrophoresis [Doktycz et al, 1995;Wunschel et al, 1996]. The Organic and Biological Mass Spectrometry Group at ORNL has developed a mass spectrometry-based method for identifying small DNA signature molecules produced by PCR amplification from natural samples.…”