Characterization of PCR Products from Bacilli Using Electrospray Ionization FTICR Mass Spectrometry

Abstract

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetronitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.

Mutation scanning by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS)

“…no need for using a DNA polymerase with 3 0 -5 0 proofreading ability, but lower productivity, to prevent the incorporation of an additional dA residue [Muddiman et al, 1996]. Using software supplied with the mass spectrometer, raw mass spectra were extracted from the reconstructed ion chromatogram for all five single strands ( Fig.…”

“…An even greater accuracy would be needed to distinguish an A4G from a T4C, or an A4C from T4G substitution as they differ by only 1 Da in mass. The use of an electrospray ionizationFourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometer, which offers the highest degree of mass resolution and mass accuracy currently attainable in mass spectrometry, would allow to scan fragments about 50% larger in size [Muddiman et al, 1996]. However, the footprint and technical expertise required for operating such instrumentation that comes at a price of about $500,000 presently excludes its use from routine mutation scanning.…”

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

“…Previous work has shown the potential of MS in bacterial genotyping. In those studies MS was used to distinguish bacterial species by size determination of PCR-amplified specific marker genes (10,11). However, for accurate bacterial identification this approach is limited, because length heterogeneities of specific marker genes do not provide sufficient discriminatory power.…”

Advances Towards Integrated Biodetection Systems for Environmental Molecular Microbiology

“…Similar instrumentation with lasers, photon counters, and sophisticated electronics can detect single genes in unamplified genomic DNA (50). While sensitivities are currently poor compared to fluorescence methods, ever more powerful mass spectrometry techniques are being rapidly developed and applied specifically for the direct detection, sequencing, and identification of nucleic acids and whole microbial cells (75)(76)(77)(78). Electrospray ionization mass spectrometry, in particular, can be fluidically linked to upstream microfluidic sample processing components (79), or integrated systems of the type described earlier.…”