DNA-SEQUENCING BY HYBRIDIZATION WITH GEL-IMMOBILIZED OLIGONUCLEOTIDES- EXPANDING APPLICABILITY WITH CHEMICAL LIGATION

Chemical ligation was used to construct a set of modified DNA duplexes containing a substituted pyrophosphate internucleotide link in a pres et position. The efficiency of chemical ligation was shown to depend o n the structure of the bond synthesis center, on thermal stability of initial DNA complexes, and on the nature of the non-nucleotide substit uent. Conditions were optimized for rapid quantitative cleavage of the substituted internucleotide bond in an aqueous medium (pH 8.0). These results can be used in DNA sequencing by hybridization with a set of oligonucleotides, which would eliminate ambiguities in determining the sequence of DNA containing repeats, and thus substantially expand the applicability of this method.