Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Progress in DNA sequence mixture readout by mass spectrometry

Chau-Wen Chou, David Dogruel, Jennifer Krone, Randall Nelson, David Schieltz and Peter Williams
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604

Mass spectrometric readout of Sanger sequencing mixtures not only has the potential to accelerate ladder readout in large-scale sequencing, but also may offer greatly reduced error rates. Faster readout may or may not accelerate the overall sequencing process, but the potential exists to keep pace with improvements in other areas, e.g. rapid end-sequence feedback for primer walking strategies. However, error elimination would produce a directly predictable rate improvement of up to a factor of 10 by eliminating the redundancy of repeated sequencing. Requirements for mass spectrometric readout include the capability to generate gas-phase molecular ions of intact DNA strands, with uniform sensitivity, mass resolution compatible with the readout length required, i.e. 1:400 for a 400 base read length, and mass spectral simplicity, i.e. one peak per DNA fragment. Large molecules can be vaporized by pulsed laser ablation from a volatile or photodegradable matrices. We are currently pursuing two approaches to this goal: (i) a search for photodegradable matrices which do not photolytically or otherwise fragment the DNA and (ii) optimization of ablation from aqueous media (ice). The best mixture spectra to date have been obtained by ablation of thin films of frozen aqueous DNA solutions, but with very poor reproducibility. Fig. 1 shows ice ablation mass spectra of the C- and G-terminated "lanes" of a synthetic sequence mixture. Favorable features of these spectra for sequencing applications are: (a) only 1 peak per DNA strand apart from some possible impurities at low mass, (b) good signal-to-noise, (c) rather uniform sensitivity (peak integrals constant within - 30% from 10--mer up to the 89-mer) (d) good registration, even though the spectra were taken some weeks apart. Missing peaks from the A and T "lanes" are immediately apparent, so that the absolute mass scale allows unambiguous error flagging, and precise mass measurement (to l 3 Da) of the 2-nucleotide gaps up to about the 40-mer allows error recovery: the identity of the missing nucleotide (A or T) can be deduced even though the A-T mass difference is only 9 Da.