Introduction to the Workshop
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Abstracts

Mapping

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Sequencing

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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.

Fragmentation Studies of Oligonucleotides Using Matrix-Assisted Laser Desorption Mass Spectrometry

Christine M. Nelson, Lin Zhu, Stephane Mouradian, Wei Tang and Lloyd M. Smith
Department of Chemistry, University of Wisconsin, Madison, WI 53706

The development of matrix-assisted laser desorption and its demonstrated performance with large proteins, up to 300,000 daltons in size, has generated substantial interest in utilizing this technique as a replacement for the gel electrophoretic separation step of the four Sanger fragment mixtures. If successful, the main advantages of this method over traditional gel-based methods are that polyacrylamide gels are no longer necessary and that the speed of separation, detection and data acquisition can be performed in seconds compared to the hours or so required in the ultrafast gel electrophoretic formats. This alternative approach is still in the preliminary stages of development. If we are able to prove its feasibility, we expect to develop an instrument with a theoretical throughput of 360,000 bases per day.

Our group has demonstrated the possibility to obtain sequencing information of oligonucleotides with 17-40 bases using the matrix 3-hydroxypicolinic acid (3HPA). In this experiment, the mass range is limited by a decrease in peak intensity for the larger components of the mixture. The current limitation of size in the MALDI analysis of oligonucleotides has generated significant interest in characterizing the problems associated with the application of this technique to nucleic acids. Early results in our laboratory and in others have shown that fragmentation is an important issue in the MALDI analysis of nucleic acids and is possibly responsible for the current limitations of size and base composition in oligonucleotide analysis.

Fragmentation studies of small oligodeoxynucleotides using MALDI-MS at 355 nm radiation from the matrix 2,5-dihydroxybenzoic acid were performed. Homopolymers of deoxythymidine are readily analyzed, however, other homopolymers and mixed sequence oligomers have been more recalcitrant. A variety of asymmetric oligonucleotides (d(T4N4T7) where N= A, C or G) were synthesized to study this fragmentation in greater detail. Similar fragmentation patterns were observed for the three samples which indicated that the primary fragmentation pathway is loss of a base followed by backbone cleavage at the 3' C-O bond of the corresponding deoxyribose. A statistical cleavage model accurately described the observed patterns of fragmentation; the model yields backbone cleavage probabilities at A, C and G of 0.13, 0.26 and 0.27, respectively.

Additional studies to further our understanding of the fragmentation mechanism have also been undertaken in which we studied the fragmentation behavior of several normal and modified DNA and RNA oligomers using MALDI, including methylated and brominated cytosine bases, abasic residues, and RNA incorporated into DNA.