Abstracts: 



Sequencing 



Technologies 



Sequencing of Megabase Plus DNA by Hybridization: 

 Method Development 



R. Crkvenjakov, R. Drmanac. Z. Strezoska. and I. Labat 



Center for Genetic Engineering, Vojvode Stepe 283. P.O. Box 283. 1 1000 Belgrade, 



Yugoslavia 



38- n -49 1-391 



The DNA sequence of a genome can be constructed by joining shorter overlapping 

 oligomer sequences of DNA. Sequencing by hybridization (SBH) is an implementation 

 in which the oligomer sequences are accessed through a DNA hybridization 

 methodology. Oligomer probes are used to ascertain the presence or absence of 

 complementary sequences in arrayed clones, with the library of overlapping clones 

 representing the genome. The sequence data gathered earlier provide for the ordering of 

 the library. The data on the unique regions of overlapping clone pairs have a particularly 

 important role and serve for the resolution of sequence branch ambiguities that would 

 otherwise arise in the developing .sequence of single clones. Through computer 

 modeling, an optimal system configuration is defined that specifies the family of 

 100,000 oligomer probes needed and the characteristics of the library. For practical 

 application, probe-template hybrids that are perfectly base-paired must be 

 distinguishable from those with a base-pair mismatch. Now that an understanding of the 

 requisite themiodynamics has been achieved and the resolving capacity demonstrated, 

 oligomers — at least as small as octamers — can serve as probes. These size reductions 

 are important because costs of oligomer synthesis are thereby greatly decreased. The 

 continuing development program includes optimization of probe labeling, library 

 cloning, and management procedures; measurement of the actual error rate in data 

 acquisition: and a stringent test of the theory. The latter is a computer simulation of data 

 processing and sequence assembly for a DNA of yeast genome size. A scheme for 

 extensive miniaturization of the probe-clone array system is under development. The 

 aim of the project is to provide experience for decision making on whether or not to 

 proceed to a sequencing pilot plant stage. 



*This project is being supported under terms of a Scientific and Technological 

 Cooperation Treaty between the United States and Yugoslavia, with Yugoslavia 

 providing the majority of the funds. 



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