Single nucleotide detection method

A method of sequencing a nucleic acid comprising the steps of:

• (a) generating a stream of single nucleotides by progressive pyrophosphorolysis of the nucleic acid;
• (b) producing at least one substantially double-stranded oligonucleotide used probe by reacting, in the presence of a polymerase and a ligase, one of the single nucleotides with a corresponding probe system comprising:
• (i) a first single-stranded oligonucleotide labelled with first and second regions of a characteristic detectable element in an undetectable state located respectively on the X′ and Y′ sides of a third region comprising a restriction enzyme recognition site element including a capture site and an exonuclease-blocking site on the X′ side thereof wherein either X′ is 3′ and Y′ is 5′, or X′ is 5′ and Y′ is 3′;
• and
• (ii) second and third single-stranded oligonucleotides that hybridize to complementary regions on the first oligonucleotide flanking the capture site;
• (c) either
• (iii) treating the used probe with a conventional or nicking substitution-dependent restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is substituted or
• (iv) treating the used probe with a conventional or nicking substitution-sensitive restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is unsubstituted;
• (d) digesting the first oligonucleotide strand of the used probe with an enzyme having double-stranded exonucleolytic activity in the X′-Y′ direction corresponding to the first oligonucleotide to yield detectable elements derived from either the first region, the second region, or the first and second regions in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide;
• (e) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe;
• (f) repeating steps (c), (d) and (e) in a cycle;
• and
• (g) detecting the detectable elements released in each iteration of step (d) wherein if the endonuclease employed is of the conventional type the second or third oligonucleotide includes an endonucleolysis-directing linkage at or close to its X′ or Y′ end respectively.