Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry

Journal Article · Fri Nov 01 04:00:00 EST 1996 · Analytical Chemistry (Washington)

DOI:https://doi.org/10.1021/ac960689j· OSTI ID:420753

Muddiman, D C; Liu, C; Pasa-Tolic, L; Anderson, G A; Smith, R D ^[1]; Wunschel, D S; Fox, K F; Fox, A ^[2]

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1. Pacific Northwest National Lab., Richland, WA (United States)
2. Univ. of South Carolina, Columbia, SC (United States)

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. 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. 45 refs., 5 figs., 1 tab.

• DOE Contract Number: AC06-76RL01830
• OSTI ID: 420753
• Journal Information: Analytical Chemistry (Washington), Journal Name: Analytical Chemistry (Washington) Journal Issue: 21 Vol. 68; ISSN ANCHAM; ISSN 0003-2700
• Country of Publication: United States
• Language: English

Related Subjects

40 CHEMISTRY
55 BIOLOGY AND MEDICINE
BASIC STUDIES
BACILLUS CEREUS
BACILLUS SUBTILIS
CHAIN REACTIONS
DNA POLYMERASES
FOURIER TRANSFORM SPECTROMETERS
ION CYCLOTRON RESONANCE SPECTROSCOPY
IONIZATION
MASS SPECTROSCOPY
NUCLEOTIDES
NUMERICAL DATA
POLYMERIZATION
RNA POLYMERASES