Investigators' Abstract): The basic objective of this project is to develop a cost effective stable sulfur isotope labeling method and automated instrument capable of determining the sequence of DNA in continuous segments longer than 1,500 bp. The method is based on: 1) an intra-chain labeling scheme which provides signal approximately independent of fragment length using four unique sulfur isotopes; 2) very high resolution physical separation of Sanger fragments using capillary gel electrophoresis; and 3) identification of the labeled fragments by continuous combustion and analysis of isotopic S(O)2 by mass spectrometry. The difficulty of reassuming fragment sequences back into genes is inversely related to the segment length. Extending the read length capability in sequencing affects the total amount of work involved in the Human Genome Project. The specific goals for research in the forthcoming period are to complete the working prototype: 1) the optimization of the physical and chemical parameters for high speed, long read DNA separations and practical methods to prepare stable, pre-cast capillary gels; 2) the development of the picoliter pump and continuous capillary electrophoresis combustion interface; 3) the design of an S(O)2 selective ionization source and mass analyzer; and 4) writing the base calling algorithms and data analysis software.
| White, D W; Drossman, H; Drayna, D (1992) A simple method for the preparation of single-stranded polydeoxynucleotides of desired length. Biotechniques 13:232-7 |
