Single nucleotide detection method and associated probes
A method of sequencing a nucleic acid comprising (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used 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 e, and at least one fluorophore region and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease; (4) digesting the first oligonucleotide component with an enzyme to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4).
1. A multi-component biological probe for capturing a single nucleoside triphosphate, the probe comprising a multiplicity of different first single-stranded oligonucleotides, wherein (a) each of the first single-stranded oligonucleotides include 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, and at least one fluorophore region located on the 5′ side of the recognition-site and arranged so as to render the fluorophore(s) quenched and (b) separate second and optionally separate third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide either side of the capture-site wherein the multiplicity of different first oligonucleotide types differs only in the nucleobase characteristic of the capture-site and the fluorophore(s); and wherein the restriction endonuclease recognition-site of the first oligonucleotide is adapted to be cleaved by a nicking endonuclease.
2. A multi-component biological probe as claimed in claim 1 , wherein the first oligonucleotide includes quencher(s) to quench the fluorophores in the fluorophore region and/or that the second oligonucleotide and third oligonucleotides are connected by an oligonucleotide linker-region.
3. A multi-component biological probe kit comprising an assembly of a multiplicity of different first oligonucleotides of the type claimed in claim 1 , each first oligonucleotide in the assembly having a different capture-site selective for a different characteristic nucleobase and a different characteristic fluorophore; wherein the kit further comprises one or more of a polymerase, a ligase, a restriction endonuclease and an enzyme having 3′-5′ exonucleolytic activity; wherein the restriction endonuclease is a nicking endonuclease adapted to cleave only the first oligonucleotide.
4. A multi-component biological probe kit as claimed in claim 3 , wherein the kit includes a third oligonucleotide.