Importance

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Abstract

The identity of a nucleotide of interest in a target nucleic acid molecule is determined by combining the target with two primers. The first primer is immobilized to a substrate and hybridizes to and extends from a location 3' of the nucleotide of interest in the target, so as to incorporate the complement of the nucleotide of interest in a first extension product. The second primer then hybridizes to and extends based on the first extension product, which is immobilized to the substrate via the first primer, at a location 3' of the complement of the nucleotide of interest, so as to incorporate the nucleotide of interest in a second extension product. The second extension product then dissociates from the first extension product and thus from the substrate and re-hybridizes to another first primer molecule that has not extended. The non-extended first primer then extends from a location 3' of the nucleotide of interest in the second extension product, so as to form, in combination with the second extension product, a double-stranded nucleic acid fragment. The first and second primers are designed to incorporate a portion of the recognition sequence of a restriction endonuclease (RE) that recognizes a partially variable interrupted nucleotide sequence, i.e., a sequence of the form D-N-S where D and S refer to specific nucleotide sequences essential for RE recognition, and N is a sequence consisting of n viable nucleotides also required for RE recognition. The first primer incorporates the sequence D, the second primer incorporates the sequence S, and they are designed, in view of the target, to product a nucleic acid fragment where constant sequences D and S are separated by variable sequence N, where the nucleotide of interest is within region N. Action of the RE on the nucleic acid fragment provides a small nucleic acid fragment that is amendable to characterization, to thereby reveal the identity of the nucleotide of interest.

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First Claim