# US 10,501,767 B2 · Polynucleotide modification methods > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,501,767 B2 - Document ID: US10501767B2 - Status: Granted - Kind: B2 - Application number: 14/911,853 - Filed: 2014-08-14 - Published: 2016-07-07 - Granted: 2019-12-10 - Earliest claimed: 2013-08-16 - Assignees: Oxford Nanopore Technologies PLC - Applicants: Oxford Nanopore Technologies Ltd. - Inventors: David Jackson Stoddart; James White - Web page: https://patentgrep.com/patent/US10501767B2 ## CPC - C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides - C12N 9/1241: {Nucleotidyltransferases (2.7.7)} - C12Q 1/68: involving nucleic acids - C12Q 1/6869: Methods for sequencing - C12Y 207/07: Nucleotidyltransferases (2.7.7) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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 - C12Y: ENZYMES ## Abstract The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterization using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterized. ## Claims ### Claim 1 A method for modifying a template double stranded polynucleotide, comprising: (a) contacting a double stranded template polynucleotide with a MuA transposase and a population of double stranded MuA substrates each comprising at least one overhang of universal nucleotides, such that the transposase fragments the template polynucleotide into two or more double-stranded fragments, and ligates a substrate to an end of each of the double stranded fragments, thereby producing a plurality of fragment/substrate constructs; and (b) ligating the overhangs to the fragments in the constructs and thereby producing a plurality of modified double stranded polynucleotides. ### Claim 2 (depends on 1) A method according to claim 1, wherein the universal nucleotide is selected from the group consisting of hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring. ### Claim 3 (depends on 2) A method according to claim 2, wherein the universal nucleotide is selected from the group consisting of 2′-deoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 4-nitroindole 2′-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2′-deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2′-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole 2′-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2′-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2′-deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2′-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5-nitroindazole 2′-deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2′-deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside or phenyl C-2′-deoxyribosyl nucleoside. ### Claim 4 (depends on 1) A method according to claim 1, wherein the overhang is five nucleotides in length. ### Claim 5 (depends on 1) A method according to claim 1, wherein (a) each substrate comprises an overhang at one end and a hairpin loop at the other end and wherein the two strands of the modified double stranded polynucleotides produced by the method are linked by the hairpin loop at one end; or (b) a proportion of the substrates in the population comprise an overhang at one end and a hairpin loop at the other end and a proportion of the substrates in the population are Y substrates with an overhang at one end and a region that is not complementary at the other end and wherein at least some of the modified double stranded polynucleotides produced by the method have the hairpin loop at one end and the non-complementary region at the other end. ### Claim 6 (depends on 1) A method according to claim 1, wherein each substrate is a Y substrate with an overhang at one end and a region that is not complementary at the other end and wherein the modified double stranded polynucleotides produced by the method have the non-complementary region at one or both ends. ### Claim 7 (depends on 1) A method according to claim 1, wherein the substrate comprises a selectable binding moiety. ### Claim 8 (depends on 5) A method according to claim 5, wherein the method further comprises separating the two strands of at least one modified double stranded polynucleotide to produce at least one single stranded polynucleotide comprising one strand of the modified double stranded polynucleotide linked to the other strand of the modified double stranded polynucleotide. ### Claim 9 (depends on 1) A method of characterising at least one polynucleotide modified using a method according to claim 1, comprising: a) contacting the modified polynucleotide with a transmembrane pore such that at least one strand of the polynucleotide moves through the pore; and b) taking one or more measurements as the at least one strand moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the at least one strand and thereby characterising the modified polynucleotide. ### Claim 10 (depends on 1) A method of characterising a template polynucleotide, comprising: a) modifying the template polynucleotide using a method according to claim 1 to produce a plurality of modified polynucleotides; b) contacting each modified polynucleotide with a transmembrane pore such that at least one strand of each polynucleotide moves through the pore; and c) taking one or more measurements as the at least one strand of each polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the at least one strand of each polynucleotide and thereby characterising the template polynucleotide. ### Claim 11 (depends on 10) A method according to claim 10, wherein the one or more characteristics are selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified. ### Claim 12 (depends on 10) A method according to claim 10, wherein step (b) further comprises contacting each modified polynucleotide with a polynucleotide binding protein such that the protein controls the movement of the at least one strand of each polynucleotide through the pore. ### Claim 13 (depends on 12) A method according to claim 12, wherein the method comprises (a) contacting each modified polynucleotide with a transmembrane pore and a polynucleotide binding protein such that at least one strand of each polynucleotide moves through the pore and the protein controls the movement of the at least one strand of each polynucleotide through the pore; and (b) measuring the current passing through the pore as the at least one strand of each polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the at least one strand of each polynucleotide and thereby characterising the template polynucleotide. ### Claim 14 (depends on 12) A method according to claim 12, wherein the polynucleotide binding protein is derived from a helicase.