Single nucleotide detection method

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

• (1) generating an ordered stream of single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid;
• (2) producing at least one double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase each single nucleoside triphosphate in the ordered stream with a probe system comprising (a) a first single-stranded oligonucleotide having a nucleoside capture region and labelled with detectable elements in an undetectable state and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide, wherein the 5′ end of the second oligonucleotide and the 3′ end of the third oligonucleotide are connected by a linker region;
• (3) digesting the used probe with an enzyme having double-stranded exonucleolytic activity to yield the detectable elements in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide;
• (4) reacting the fourth oligonucleotide with another molecule of the first oligonucleotide of the probe system to produce a double-stranded oligonucleotide product corresponding to the used probe;
• (5) repeating steps (3) and (4) in a cycle and
• (6) detecting the detectable elements released in each iteration of step (3).