# US 2010/0167280 A1 · Oligonucleotides for discriminating related nucleic acid sequences > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2010/0167280 A1 - Document ID: US20100167280A1 - Status: Published application - Kind: A1 - Application number: 12/377,044 - Filed: 2007-08-10 - Published: 2010-07-01 - Earliest claimed: 2006-08-11 - Inventors: Ivan Brukner; Damian Labuda; Maja Krajinovic - Web page: https://patentgrep.com/patent/US20100167280A1 ## CPC - C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules - C12Q 1/708: {for papilloma} - C12N 2710/20011: Papillomaviridae - 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 - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract An in vitro selection method is described which identifies oligonucleotide probes that discriminate amongst closely related nucleic acid sequences and which involves iterative hybridizations, including subtractive hybridization. Using the method, oligonucleotides are identified which can discriminate among human papilloma virus (HPV) subtypes. Corresponding methods and kits for the detection of nucleic acids are described, which methods and kits may be used in analytical, diagnostic, research and related applications. ## Claims ### Claim 1 A method for identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, said method comprising: (a) hybridizing said first nucleic acid with a pool of oligonucleotides in a hybridization mixture, said oligonucleotides comprising a random nucleotide sequence flanked by primer recognition sequences; (b) removing oligonucleotides which are not bound to said first nucleic acid from said hybridization mixture; (c) dissociating bound oligonucleotides from said first nucleic acid; (d) amplifying said bound oligonucleotides using primers capable of binding to said primer recognition sequences to obtain amplified oligonucleotide duplexes comprising a first strand corresponding to said bound oligonucleotides and a second strand corresponding to the complement of said bound oligonucleotides; (e) treating said duplexes to remove or degrade said second strand to obtain single-stranded amplified oligonucleotides; (f) repeating (a) to (e), wherein said pool of oligonucleotides of (a) is the amplified oligonucleotides obtained in (e) thereby to obtain further amplified oligonucleotides; and (g) repeating (a) to (e), wherein said hybridization step (a) is performed in the further presence of said second nucleic acid; wherein an oligonucleotide comprising the random nucleotide sequence of said further amplified oligonucleotides can be used for discriminating said first nucleic acid from said second nucleic acid. ### Claim 2 (depends on 1) The method according to claim 1, wherein said repeating step (f) is performed at least 2 times. ### Claim 3 (depends on 2) The method according to claim 2, wherein said repeating step (f) is performed at least 4 times. ### Claim 4 (depends on 1, 2, 3) The method according to any one of claims 1 to 3, wherein said repeating step (g) is performed at least 2 times. ### Claim 5 (depends on 4) The method according to claim 4, wherein said repeating step (g) is performed at least 3 times. ### Claim 6 (depends on 1, 2, 3, 4, 5) The method according to any one of claims 1 to 5, further comprising selecting an oligonucleotide from said further amplified oligonucleotides on the basis of its preferential binding to said first nucleic acid relative to said second nucleic target. ### Claim 7 (depends on 1, 2, 3, 4, 5, 6) The method according to any one of claims 1 to 6, wherein said hybridization is performed in the presence of a blocking agent capable of inhibiting binding of said primer recognition sequences to said first nucleic acid. ### Claim 8 (depends on 7) The method according to claim 7 wherein said blocking agent is an oligonucleotide capable of binding said primer recognition sequences. ### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The method according to any one of claims 1-8, wherein said first nucleic acid is derived from a pathogen. ### Claim 10 (depends on 9) The method according to claim 9, wherein said pathogen is selected from a eukaryote, prokaryote and a virus. ### Claim 11 (depends on 10) The method according to claim 10, wherein said virus is human papillomavirus (HPV). ### Claim 12 (depends on 11) The method according to claim 11, wherein said first and second nucleic acids are derived from different subtypes of HPV. ### Claim 13 (depends on 12) The method according to claim 12, wherein said subtypes are selected from HPV 6, HPV 11, HPV 13, HPV 16, HPV 18, HPV 26, HPV 30, HPV 31, HPV 33, HPV 34, HPV 35, HPV 39, HPV 40, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 62, HPV 64, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 72, HPV 73, HPV 74, HPV MM4, HPV MM7 and HPV MM8. ### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) The method according to any one of claims 1-13, wherein said first nucleic acid is bound to a solid support. ### Claim 15 (depends on 14) The method according to claim 14, wherein the random nucleotide sequence of said further amplified oligonucleotides comprises at least 1 mismatch relative to said first nucleic acid. ### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) The method according to any one of claims 1-15, wherein said amplification is performed using polymerase chain reaction (PCR) or isothermal amplification. ### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) The method according to any one of claims 1-16, wherein said dissociation is performed by incubation at an elevated temperature relative to said hybridization. ### Claim 18 (depends on 17) The method according to claim 17, wherein said elevated temperature is a temperature above the thermal melting point (Tm). ### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The method according to any one of claims 1 to 18, wherein said treatment is with an exonuclease capable of selective degradation of said second strand. ### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) An oligonucleotide identified by the method according to any one of claims 1-19. ### Claim 21 An oligonucleotide capable of discriminating a first nucleic acid from a second nucleic acid, wherein said oligonucleotide is not exactly complementary to said first nucleic acid. ### Claim 22 (depends on 21) The oligonucleotide according to claim 21, wherein said oligonucleotide comprises at least 1 mismatch relative to said first nucleic acid. ### Claim 23 (depends on 21, 22) The oligonucleotide according to claim 21 or 22, wherein said first nucleic acid is derived from a pathogen. ### Claim 24 (depends on 23) The oligonucleotide according to claim 23, wherein said pathogen is selected from a eukaryote, prokaryote and a virus. ### Claim 25 (depends on 24) The oligonucleotide according to claim 24, wherein said virus is human papillomavirus (HPV). ### Claim 26 (depends on 25) The oligonucleotide according to claim 25, wherein said first and second nucleic acids are derived from different subtypes of HPV. ### Claim 27 (depends on 26) The oligonucleotide according to claim 26, wherein said subtypes are selected from HPV 6, HPV 11, HPV 13, HPV 16, HPV 18, HPV 26, HPV 30, HPV 31, HPV 33, HPV 34, HPV 35, HPV 39, HPV 40, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 62, HPV 64, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 72, HPV 73, HPV 74, HPV MM4, HPV MM7 and HPV MM8. ### Claim 28 (depends on 27) The oligonucleotide according to claim 27, comprising a nucleotide sequence selected from SEQ ID NOs: 1-43, 100-104 and 116. ### Claim 29 (depends on 28) The oligonucleotide according to claim 28, wherein said oligonucleotide comprises a sequence and is capable of selectively detecting an HPV subtype as set forth in Table I. ### Claim 30 (depends on 28) The probe according to claim 28 wherein said oligonucleotide is selected from: an oligonucleotide comprising SEQ ID NO: 1, 2, 100 or 116 and which is capable of selectively detecting HPV 6; an oligonucleotide comprising SEQ ID NO: 3, 4 or 101 and which is capable of selectively detecting HPV 11; an oligonucleotide comprising SEQ ID NO: 5 and which is capable of selectively detecting HPV 13; an oligonucleotide comprising SEQ ID NO: 6 or 102 and which is capable of selectively detecting HPV 16; an oligonucleotide comprising SEQ ID NO: 7 or 103 and which is capable of selectively detecting HPV 18; an oligonucleotide comprising SEQ ID NO: 8 and which is capable of selectively detecting HPV 26; an oligonucleotide comprising SEQ ID NO: 9 and which is capable of selectively detecting HPV 30; an oligonucleotide comprising SEQ ID NO: 10 and which is capable of selectively detecting HPV 31; an oligonucleotide comprising SEQ ID NO: 11 and which is capable of selectively detecting HPV 33; an oligonucleotide comprising SEQ ID NO: 12 and which is capable of selectively detecting HPV 34; an oligonucleotide comprising SEQ ID NO: 13 and which is capable of selectively detecting HPV 39; an oligonucleotide comprising SEQ ID NO: 14, 15 or 16 and which is capable of selectively detecting HPV 39; an oligonucleotide comprising SEQ ID NO: 17 and which is capable of selectively detecting HPV 40; an oligonucleotide comprising SEQ ID NO: 18 and which is capable of selectively detecting HPV 42; an oligonucleotide comprising SEQ ID NO: 19 and which is capable of selectively detecting HPV 43; an oligonucleotide comprising SEQ ID NO: 20 and which is capable of selectively detecting HPV 44; an oligonucleotide comprising SEQ ID NO: 21 and which is capable of selectively detecting HPV 45; an oligonucleotide comprising SEQ ID NO: 22 and which is capable of selectively detecting HPV 51; an oligonucleotide comprising SEQ ID NO: 23 and which is capable of selectively detecting HPV 52; an oligonucleotide comprising SEQ ID NO: 24 and which is capable of selectively detecting HPV 53; an oligonucleotide comprising SEQ ID NO: 25 and which is capable of selectively detecting HPV 54; an oligonucleotide comprising SEQ ID NO: 26 and which is capable of selectively detecting HPV 55; an oligonucleotide comprising SEQ ID NO: 27 and which is capable of selectively detecting HPV 56; an oligonucleotide comprising SEQ ID NO: 28 and which is capable of selectively detecting HPV 58; an oligonucleotide comprising SEQ ID NO: 29 and which is capable of selectively detecting HPV 59; an oligonucleotide comprising SEQ ID NO: 30 and which is capable of selectively detecting HPV 61; an oligonucleotide comprising SEQ ID NO: 31 and which is capable of selectively detecting HPV 62; an oligonucleotide comprising SEQ ID NO: 32 and which is capable of selectively detecting HPV 64; an oligonucleotide comprising SEQ ID NO: 33 and which is capable of selectively detecting HPV 66; an oligonucleotide comprising SEQ ID NO: 34 and which is capable of selectively detecting HPV 67; an oligonucleotide comprising SEQ ID NO: 35 and which is capable of selectively detecting HPV 68; an oligonucleotide comprising SEQ ID NO: 36 and which is capable of selectively detecting HPV 69; an oligonucleotide comprising SEQ ID NO: 37 and which is capable of selectively detecting HPV 70; an oligonucleotide comprising SEQ ID NO: 38 and which is capable of selectively detecting HPV 72; an oligonucleotide comprising SEQ ID NO: 39 and which is capable of selectively detecting HPV 73; an oligonucleotide comprising SEQ ID NO: 40 and which is capable of selectively detecting HPV 74; an oligonucleotide comprising SEQ ID NO: 41 and which is capable of selectively detecting HPV MM4; an oligonucleotide comprising SEQ ID NO: 42 and which is capable of selectively detecting HPV MM7; an oligonucleotide comprising SEQ ID NO: 43 and which is capable of selectively detecting HPV MM8; and an oligonucleotide comprising SEQ ID NO: 104 and which is capable of selectively detecting HPV 31 and/or 33. ### Claim 31 An oligonucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 1-43, 100-104 and 116. ### Claim 32 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) A method for detecting the presence or absence of a first nucleic acid in a sample, said method comprising contacting the oligonucleotide according to any one of claims 20-31 with said sample under conditions permitting selective hybridization of said oligonucleotide to said first nucleic acid, wherein said selective hybridization is indicative that said first nucleic acid is present in said sample. ### Claim 33 (depends on 32) The method according to claim 32, wherein said first nucleic acid is derived from a pathogen and said method is for detection of said pathogen in a sample. ### Claim 34 (depends on 33) The method according to claim 33, wherein said sample is a biological sample derived from a subject and said method is for detection of said pathogen in said subject. ### Claim 35 (depends on 34) The method according to claim 34, wherein said method is for diagnosing a disease or condition associated with said pathogen in said subject. ### Claim 36 (depends on 33, 34, 35) The method according to any one of claims 33-35, wherein said pathogen is selected from a eukaryote, prokaryote and a virus. ### Claim 37 (depends on 36) The method according to claim 36, wherein said virus is human papillomavirus (HPV). ### Claim 38 (depends on 37) The method according to claim 37, wherein said method is for detecting the presence of a subtype of HPV. ### Claim 39 (depends on 38) The method according to claim 38, wherein said subtype is selected from HPV 6, HPV 11, HPV 13, HPV 16, HPV 18, HPV 26, HPV 30, HPV 31, HPV 33, HPV 34, HPV 35, HPV 39, HPV 40, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 62, HPV 64, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 72, HPV 73, HPV 74, HPV MM4, HPV MM7 and HPV MM8. ### Claim 40 (depends on 39) The method according to claim 39, wherein said oligonucleotide is selected from: an oligonucleotide comprising SEQ ID NO: 1, 2, 100 or 116 and which is capable of selectively detecting HPV 6; an oligonucleotide comprising SEQ ID NO: 3, 4 or 101 and which is capable of selectively detecting HPV 11; an oligonucleotide comprising SEQ ID NO: 5 and which is capable of selectively detecting HPV 13; an oligonucleotide comprising SEQ ID NO: 6 or 102 and which is capable of selectively detecting HPV 16; an oligonucleotide comprising SEQ ID NO: 7 or 103 and which is capable of selectively detecting HPV 18; an oligonucleotide comprising SEQ ID NO: 8 and which is capable of selectively detecting HPV 26; an oligonucleotide comprising SEQ ID NO: 9 and which is capable of selectively detecting HPV 30; an oligonucleotide comprising SEQ ID NO: 10 and which is capable of selectively detecting HPV 31, an oligonucleotide comprising SEQ ID NO: 11 and which is capable of selectively detecting HPV 33; an oligonucleotide comprising SEQ ID NO: 12 and which is capable of selectively detecting HPV 34; an oligonucleotide comprising SEQ ID NO: 13 and which is capable of selectively detecting HPV 39; an oligonucleotide comprising SEQ ID NO: 14, 15 or 16 and which is capable of selectively detecting HPV 39; an oligonucleotide comprising SEQ ID NO: 17 and which is capable of selectively detecting HPV 40; an oligonucleotide comprising SEQ ID NO: 18 and which is capable of selectively detecting HPV 42; an oligonucleotide comprising SEQ ID NO: 19 and which is capable of selectively detecting HPV 43; an oligonucleotide comprising SEQ ID NO: 20 and which is capable of selectively detecting HPV 44; an oligonucleotide comprising SEQ ID NO: 21 and which is capable of selectively detecting HPV 45; an oligonucleotide comprising SEQ ID NO: 22 and which is capable of selectively detecting HPV 51; an oligonucleotide comprising SEQ ID NO: 23 and which is capable of selectively detecting HPV 52; an oligonucleotide comprising SEQ ID NO: 24 and which is capable of selectively detecting HPV 53; an oligonucleotide comprising SEQ ID NO: 25 and which is capable of selectively detecting HPV 54; an oligonucleotide comprising SEQ ID NO: 26 and which is capable of selectively detecting HPV 55; an oligonucleotide comprising SEQ ID NO: 27 and which is capable of selectively detecting HPV 56; an oligonucleotide comprising SEQ ID NO: 28 and which is capable of selectively detecting HPV 58; an oligonucleotide comprising SEQ ID NO: 29 and which is capable of selectively detecting HPV 59; an oligonucleotide comprising SEQ ID NO: 30 and which is capable of selectively detecting HPV 61; an oligonucleotide comprising SEQ ID NO: 31 and which is capable of selectively detecting HPV 62; an oligonucleotide comprising SEQ ID NO: 32 and which is capable of selectively detecting HPV 64; an oligonucleotide comprising SEQ ID NO: 33 and which is capable of selectively detecting HPV 66; an oligonucleotide comprising SEQ ID NO: 34 and which is capable of selectively detecting HPV 67; an oligonucleotide comprising SEQ ID NO: 35 and which is capable of selectively detecting HPV 68; an oligonucleotide comprising SEQ ID NO: 36 and which is capable of selectively detecting HPV 69; an oligonucleotide comprising SEQ ID NO: 37 and which is capable of selectively detecting HPV 70; an oligonucleotide comprising SEQ ID NO: 38 and which is capable of selectively detecting HPV 72; an oligonucleotide comprising SEQ ID NO: 39 and which is capable of selectively detecting HPV 73; an oligonucleotide comprising SEQ ID NO: 40 and which is capable of selectively detecting HPV 74; an oligonucleotide comprising SEQ ID NO: 41 and which is capable of selectively detecting HPV MM4; an oligonucleotide comprising SEQ ID NO: 42 and which is capable of selectively detecting HPV MM7; an oligonucleotide comprising SEQ ID NO: 43 and which is capable of selectively detecting HPV MM8; and an oligonucleotide comprising SEQ ID NO: 104 and which is capable of selectively detecting HPV 31 and/or 33. ### Claim 41 (depends on 34, 36) The method according to claim 34 or 36, wherein said subject is a mammal. ### Claim 42 (depends on 41) The method according to claim 41, wherein said mammal is a human. ### Claim 43 (depends on 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) The method according to any one of claims 32-42, wherein said oligonucleotide is bound to a solid support. ### Claim 44 (depends on 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) The method according to any one of claims 32-43, wherein said first nucleic acid is labelled with a detectable marker. ### Claim 45 (depends on 44) The method according to claim 44, wherein said detectable marker is a fluorescent moiety. ### Claim 46 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) A kit for detecting the presence of a first nucleic acid in a sample, said kit comprising the oligonucleotide according to any one of claims 20-31. ### Claim 47 (depends on 46) The kit of claim 46, further comprising means for detecting selective hybridization of said oligonucleotide to said first nucleic acid. ### Claim 48 (depends on 31, 46, 47) The kit according to claim 46 or 47, further comprising instructions setting forth the method of claim 31. ### Claim 49 (depends on 46, 47, 48) The kit according to any one of claims 46 to 48, wherein said first nucleic acid is derived from a pathogen and said kit is for detecting the presence of said pathogen in said sample. ### Claim 50 (depends on 49) The kit according to claim 49, wherein said sample is a biological sample derived from a subject and said kit is for detection of said pathogen in said subject. ### Claim 51 (depends on 50) The kit according to claim 50, wherein said kit is for diagnosing a disease or condition associated with said pathogen in said subject. ### Claim 52 (depends on 48, 49, 50, 51) The kit according to any one of claims 48-51, wherein said pathogen is selected from a eukaryote, prokaryote and a virus. ### Claim 53 (depends on 52) The kit according to claim 52, wherein said virus is human papillomavirus (HPV). ### Claim 54 (depends on 53) The kit according to claim 53, wherein said kit is for detecting the presence of a subtype of HPV. ### Claim 55 (depends on 54) The kit according to claim 54, wherein said subtype is selected from HPV 6, HPV 11, HPV 13, HPV 16, HPV 18, HPV 26, HPV 30, HPV 31, HPV 33, HPV 34, HPV 35, HPV 39, HPV 40, HPV 42, HPV 43, HPV 44, HPV 45, HPV 51, HPV 52, HPV 53, HPV 54, HPV 55, HPV 56, HPV 58, HPV 59, HPV 61, HPV 62, HPV 64, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 72, HPV 73, HPV 74, HPV MM4, HPV MM7 and HPV MM8. ### Claim 56 (depends on 30, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55) The kit according to any one of claims 46-55, wherein said oligonucleotide is the oligonucleotide of claim 30. ### Claim 57 A collection of two or more oligonucleotides, wherein said oligonucleotides comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-43, 100-104 and 116. ### Claim 58 (depends on 57) The collection according to claim 57, wherein said oligonucleotides are immobilized on a substrate. ### Claim 59 (depends on 57, 58) The collection of any one of claims 57-58, wherein said oligonucleotides are hybridizable array elements in a microarray. ### Claim 60 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 57, 58, 59) An array comprising the oligonucleotide according to any one of claims 20-31 or the collection of two or more oligonucleotides according to any one of claims 57-59. ### Claim 61 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) A kit for identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, said kit comprising the pool of oligonucleotides defined in any one of claims 1-19. ### Claim 62 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 61) The kit according to claim 61, further comprising instructions setting forth the method according to any one of claims 1-19.