SINGLE NUCLEOTIDE DETECTION METHOD AND ASSOCIATED PROBES

A method of sequencing a nucleic acid comprising the steps of (1) generating a stream of single nucleoside triphosphates; (2) producing at least one substantially double-stranded primary oligonucleotide used probe by reacting with a corresponding primary probe comprising (a) a first single-stranded oligonucleotide including a restriction endonuclease nicking-site, a single nucleotide capture site, and oligonucleotide flanking regions. and (b) second and third single-stranded oligonucleotides; (3) nicking the first oligonucleotide strand of the used primary probe to create separate first oligonucleotide components; (4) separating the first oligonucleotide components from the complementary strand of the used probe; (5) producing at least one substantially double-stranded secondary used probe by reacting, with a corresponding secondary probe comprising (c) a complementary fourth oligonucleotide comprising fluorophores; (6) digesting the used secondary probe with an enzyme to yield the fluorophores; and a single-stranded sixth oligonucleotide and (7) detecting the fluorophores released in step (6).