# US 10,480,024 B2 · Single nucleotide detection method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,480,024 B2 - Document ID: US10480024B2 - Status: Granted - Kind: B2 - Application number: 14/782,650 - Filed: 2014-04-09 - Published: 2016-02-11 - Granted: 2019-11-19 - Earliest claimed: 2014-04-09 - Assignees: Base4 Innovation Ltd; United Kingdom Research and Innovation - Applicants: Base4 Innovation Ltd; Medical Research Council - Inventors: Cameron Alexander Frayling; Barnaby Balmforth; Bruno Flavio Nogueira de Sousa Soares; Thomas Henry Isaac; Boris Breiner; Alessandra Natale; Michele Amasio; Paul Dear - Web page: https://patentgrep.com/patent/US10480024B2 ## CPC - C12Q 1/6869: Methods for sequencing - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system; (3) amplifying at least part of the captured molecule to produce a plurality of amplicons characteristic of the single nucleotide base; (4) labelling the amplicons with a corresponding probe having a characteristic detectable element and (5) detecting a property characteristic of the detectable element. ## Claims ### Claim 1 A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of: (1) generating a stream of single nucleotide bases from the polynucleotide analyte, wherein the stream of single nucleotide bases is obtained by progressive pyrophosphorolysis of the polynucleotide analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base; (3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons; (4) for each captured molecule, labelling the amplicons with a corresponding probe selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base; (5) for each captured molecule, detecting a property characteristic of the detectable element; and (6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte. ### Claim 2 (depends on 1) The method as claimed in claim 1, wherein the capture system is comprised of two components for each different nucleotide base: (a) a first oligonucleotide comprising a double-stranded region and a single-stranded region and (b) a second oligonucleotide that is single-stranded and whose nucleotide base sequence is at least partially complementary to that of the single-stranded region of the first oligonucleotide one nucleotide base beyond the end of the double-stranded region. ### Claim 3 (depends on 2) The method as claimed in claim 2, wherein the first oligonucleotide is j-shaped. ### Claim 4 (depends on 2) The method as claimed in claim 2, wherein the total length of the first oligonucleotide is from 20 to 100 nucleotide bases. ### Claim 5 (depends on 1) The method as claimed in claim 1, wherein the capture system comprises a single oligonucleotide for each different nucleotide base, consisting of a single-stranded nucleotide region the ends of which are attached to two different double-stranded oligonucleotide regions. ### Claim 6 (depends on 5) The method as claimed in claim 5, wherein each double-stranded oligonucleotide region is comprised of from 10 to 30 nucleotide pairs. ### Claim 7 (depends on 5) The method as claimed in claim 5, wherein two discrete double-stranded oligonucleotide regions are employed, each comprising ends remote from the single-stranded nucleotide region which are closed-looped. ### Claim 8 (depends on 5) The method as claimed in claim 5, wherein the double-stranded oligonucleotide regions are derivable from a single-stranded oligonucleotide precursor by folding the ends back on themselves to leave a gap comprising the single-stranded nucleotide region. ### Claim 9 (depends on 1) The method as claimed in claim 1, wherein the polynucleotide analyte is bound to a surface. ### Claim 10 (depends on 1) The method as claimed in claim 1, wherein step (1) is carried out under non-equilibrium conditions in the presence of a flowing aqueous medium comprising an enzyme, a pyrophosphate anion and magnesium cations, and wherein the single nucleotide bases are continuously removed from a reaction zone where they are generated. ### Claim 11 (depends on 10) The method as claimed in claim 10, wherein between steps (1) and (2), any residual pyrophosphate anion is destroyed by means of a pyrophosphatase. ### Claim 12 (depends on 1) The method as claimed in claim 1, wherein the capture system comprises four different first oligonucleotides, each of which has one of four different single-stranded regions, and four different second oligonucleotides, each of which has a sequence complementary to a part of one of the four different single-stranded regions in the four different first oligonucleotides. ### Claim 13 (depends on 1) The method as claimed in claim 1, wherein steps (3) and (4) are carried out simultaneously. ### Claim 14 (depends on 1) The method as claimed in claim 1, wherein the amplification in step (3) is carried out using a method selected from the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification. ### Claim 15 (depends on 1) The method as claimed in claim 1, wherein step (2) employs a ligase which is deactivated before step (4) occurs. ### Claim 16 (depends on 1) The method as claimed in claim 1, wherein the probes are molecular beacons. ### Claim 17 (depends on 1) The method as claimed in claim 1, wherein in step (5) the detectable element is a fluorophore and fluorescence emitted by fluorophores is detected. ### Claim 18 (depends on 1) The method as claimed in claim 1, wherein at least one of steps (1) to (5) is carried out in microdroplets. ### Claim 19 A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of: (1) generating a stream of single nucleotide bases from the polynucleotide analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base, and wherein the capture system is comprised of two components for each different nucleotide base: (a) a first oligonucleotide comprising a double-stranded region and a single-stranded region and (b) a second oligonucleotide that is single-stranded and whose nucleotide base sequence is at least partially complementary to that of the single-stranded region of the first oligonucleotide one nucleotide base beyond the end of the double-stranded region; (3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons; (4) for each captured molecule, labelling the amplicons with a corresponding probe that is selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base; (5) for each captured molecule, detecting a property characteristic of the detectable element; and (6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte. ### Claim 20 (depends on 19) The method as claimed in claim 19, wherein the stream of single nucleotide bases in step (1) is obtained by action of (i) a kinase and (ii) an exonuclease. ### Claim 21 (depends on 19) The method as claimed in claim 19, wherein the polynucleotide analyte is bound to a surface. ### Claim 22 (depends on 19) The method as claimed in claim 19, wherein step (1) is carried out under non-equilibrium conditions in the presence of a flowing aqueous medium comprising an enzyme, a pyrophosphate anion and magnesium cations, and wherein the single nucleotide bases are continuously removed from a reaction zone where they are generated. ### Claim 23 (depends on 22) The method as claimed in claim 22, wherein between steps (1) and (2), any residual pyrophosphate anion is destroyed by means of a pyrophosphatase. ### Claim 24 (depends on 19) The method as claimed in claim 19, wherein the first oligonucleotide is j-shaped. ### Claim 25 (depends on 19) The method as claimed in claim 19, wherein the total length of the first oligonucleotide is from 20 to 100 nucleotide bases. ### Claim 26 (depends on 19) The method as claimed in claim 19, wherein the polynucleotide analyte is DNA or RNA, and the capture system comprises four different sets of capture molecules, each set being selective for a different nucleotide base of the DNA or RNA. ### Claim 27 (depends on 19) The method as claimed in claim 19, wherein steps (3) and (4) are carried out simultaneously. ### Claim 28 (depends on 19) The method as claimed in claim 19, wherein the amplification in step (3) is carried out using a method selected from the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification. ### Claim 29 (depends on 19) The method as claimed in claim 19, wherein step (2) employs a ligase which is deactivated before step (4) occurs. ### Claim 30 (depends on 19) The method as claimed in claim 19, wherein the probes are molecular beacons. ### Claim 31 (depends on 19) The method as claimed in claim 19, wherein in step (5) the detectable element is a fluorophore and fluorescence emitted by fluorophores is detected. ### Claim 32 (depends on 19) The method as claimed in claim 19, wherein at least one of steps (1) to (5) is carried out in microdroplets. ### Claim 33 A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of: (1) generating a stream of single nucleotide bases from the polynucleotide analyte, wherein step (1) is carried out in the presence of a reaction medium comprising a polymerase that exhibits neither exonuclease or endonuclease behaviour under conditions of a pyrophosphorolysis reaction; (2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base; (3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons; (4) for each captured molecule, labelling the amplicons with a corresponding probe that is selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base; (5) for each captured molecule, detecting a property characteristic of the detectable element; and (6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte. ### Claim 34 (depends on 33) The method as claimed in claim 33, wherein the capture system is comprised of two components for each different nucleotide base: (a) a first oligonucleotide comprising a double-stranded region and a single-stranded region and (b) a second oligonucleotide that is single-stranded and whose nucleotide base sequence is at least partially complementary to that of the single-stranded region of the first oligonucleotide one nucleotide base beyond the end of the double-stranded region. ### Claim 35 (depends on 34) The method as claimed in claim 34, wherein the first oligonucleotide is j-shaped. ### Claim 36 (depends on 34) The method as claimed in claim 34, wherein the total length of the first oligonucleotide is from 20 to 100 nucleotide bases. ### Claim 37 (depends on 33) The method as claimed in claim 33, wherein the capture system comprises a single oligonucleotide for each different nucleotide base, consisting of a single-stranded nucleotide region the ends of which are attached to two different double-stranded oligonucleotide regions. ### Claim 38 (depends on 37) The method as claimed in claim 37, wherein each double-stranded oligonucleotide region is comprised of from 10 to 30 nucleotide pairs. ### Claim 39 (depends on 37) The method as claimed in claim 37, wherein two discrete double-stranded oligonucleotide regions are employed, each comprising ends remote from the single-stranded nucleotide region which are closed-looped. ### Claim 40 (depends on 37) The method as claimed in claim 37, wherein the double-stranded oligonucleotide regions are derivable from a single-stranded oligonucleotide precursor by folding the ends back on themselves to leave a gap comprising the single-stranded nucleotide region. ### Claim 41 (depends on 33) The method as claimed in claim 33, wherein the polynucleotide analyte is bound to a surface. ### Claim 42 (depends on 33) The method as claimed in claim 33, wherein step (1) is carried out under non-equilibrium conditions in the presence of a flowing aqueous medium comprising the polymerase, a pyrophosphate anion and magnesium cations, and wherein the single nucleotide bases are continuously removed from a reaction zone where they are generated. ### Claim 43 (depends on 42) The method as claimed in claim 42, wherein between steps (1) and (2), any residual pyrophosphate anion is destroyed by means of a pyrophosphatase. ### Claim 44 (depends on 33) The method as claimed in claim 33, wherein the polynucleotide analyte is DNA or RNA, and the capture system comprises four different sets of capture molecules, each set being selective for a different nucleotide base of the DNA or RNA. ### Claim 45 (depends on 33) The method as claimed in claim 33, wherein steps (3) and (4) are carried out simultaneously. ### Claim 46 (depends on 33) The method as claimed in claim 33, wherein the amplification in step (3) is carried out using a method selected from the polymerase chain reaction, recombinase polymerase amplification and rolling circle amplification. ### Claim 47 (depends on 33) The method as claimed in claim 33, wherein step (2) employs a ligase which is deactivated before step (4) occurs. ### Claim 48 (depends on 33) The method as claimed in claim 33, wherein the probes are molecular beacons. ### Claim 49 (depends on 33) The method as claimed in claim 33, wherein in step (5) the detectable element is a fluorophore and fluorescence emitted by fluorophores is detected. ### Claim 50 (depends on 33) The method as claimed in claim 33, wherein at least one of steps (1) to (5) is carried out in microdroplets.