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 by progressive enzymatic digestion of the nucleic acid;
• (2) producing at least one used oligonucleotide probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5′ side of the blocking-site and including a single nucleotide capture-site for capturing the single nucleoside triphosphate, and at least one fluorophore region located on the 5′ side of the recognition-site comprising fluorophore(s) arranged so as to render the fluorophore(s) quenched, wherein the fluorophore(s) are arranged in close proximity to one another in order to self-quench or wherein the at least one fluorophore region further comprises a quencher configured to quench the fluorophore(s), and (b) a second and a third single-stranded oligonucleotide each separate from the first oligonucleotide and capable of hybridising to complementary regions on the first oligonucleotide flanking the 3′ and 5′ sides of the capture-site;
• (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease to create a first oligonucleotide component bearing the fluorophore(s);
• (4) digesting the first oligonucleotide component with an enzyme having 3′-5′ exonucleolytic activity to yield fluorophore(s) in a detectable state;
• and
• (5) detecting the fluorophore(s) released in step (4);
• wherein the restriction endonuclease is a nicking endonuclease adapted to cleave only the first oligonucleotide;
• and
• wherein steps (2) and (3) are iterated and step (4) is carried out after the iteration.