|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
44. DNA Characterization by Electrospray Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
David S. Wunschel, Ljiljana Pasa Tolic,
Bingbing Feng, James E. Bruce, Harold R. Udseth, and Richard D. Smith
Mass spectrometry offers the potential for high speed DNA sequencing and ultra-sensitive characterization. Ongoing work in the laboratory is exploring approaches based upon electrospray ionization (ESI) and/or Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. These efforts have included advanced methods for the characterization of polymerase chain reaction (PCR) products 1, enzymatically produced oligonucleotide mixtures, modified DNA and the development of methods for the analysis of DNA large fragments. High mass accuracy measurements for PCR products allowing a single base substitutions to be detected at the 250 bp level with de novo identification of an unreported base substitution. This capability also allows the identification of small differences in mass such as those arising from methylation2. Study of DNA damage/modifications in their sequence context will likely have to occur from within multi-component mixtures. The capability for this has been demonstrated using a multi-component reaction where a base pair deletion was identified with the putative identification of inter-operon variability within a single bacterial strain3. These efforts are also being extended to exploit the non-destructive nature of FTICR for recovery (i.e., "soft-landing") of mass-selected modified DNA segments, following high resolution FTICR analysis and separation (i.e., high resolution sorting), for subsequent cloning or PCR. This would allow for direct selection and analysis of individual components from within mixtures that may share a high degree of similarity without cloning. Alternatively, DNA species that cannot be identified through traditional sequencing methodologies, those containing base modifications, can be isolated with the nature and position of the modification identified. Most importantly this potentially allow identification of low abundance products containing modifications where few if any alternatives for their detection exist. These and related recent advances will be described.
1 "Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry", D. C. Muddiman, D. S. Wunschel, C. L. Liu, L. Pasa Tolic, K. F. Fox, A. Fox, G. A. Anderson and R. D. Smith, Anal. Chem. 68, 3705-3712 (1996)
2 "Mass measurement of a PCR product from the Lambda bacteria phage at the 223 base pair level by ESI-FTICR", D. S. Wunschel, B. Feng, L. Pasa Tolic, and R. D. Smith.
3 "Heterogeneity in Bacillus cereus PCR Products Detected by ESI-FTICR Mass Spectrometry", D. S. Wunschel, D. C. Muddiman, K. F. Fox, A. Fox, and R. D. Smith, Anal. Chem. 70, 1203-1207 (1998)
This research was supported by the Office of Biological and Environmental Research, U.S. Department of Energy. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute through Contract No. DE-AC06-76RLO 1830.
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