Amplification of target nucleic acids using gap filling ligase chain reaction
First Claim
1. A method for detecting the presence of a target nucleic acid in a sample, said method employing a ligase chain reaction to create geometrically increasing numbers of reorganized probe molecules in the presence of said target nucleic acid, said method comprising:
- a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula;
5'"'"'-(N)h XEp Fq Z(N)k -3'"'"' or its complement 3'"'"'-(N'"'"')h X'"'"'E'"'"'p F'"'"'q Z'"'"'(N'"'"')k -5'"'"', wherein E represents any base, E'"'"' is the complement of E, F represents any base except E, F'"'"' is the complement of F, p and q are independently integers from 1 to about 10, X represents any base except E or F'"'"', X'"'"' is the complement of X, Z represents any base except F or E'"'"', Z'"'"'is the complement of Z, N represents any base, and h and k are independently integers from 5 to 100, provided (p+h) and (q+k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected;
b) providing a plurality of each of four probes having the formulas;
space="preserve" listing-type="equation">5'"'"'-(N).sub.h X E.sub.p -3'"'"' A
space="preserve" listing-type="equation">3'"'"'-(N'"'"').sub.h X'"'"'-5'"'"' A'"'"'
space="preserve" listing-type="equation">5'"'"'-Z(N).sub.k -3'"'"' B
space="preserve" listing-type="equation">3'"'"'-F'"'"'.sub.q Z'"'"'(N'"'"').sub.k -5'"'"'wherein N, N'"'"', X, X'"'"', Z, Z'"'"'E, F'"'"', h, p, k and q are defined above, except that A and A'"'"' or B and B'"'"' need not be identical in length, and wherein at least one of the probes is labeled with a moiety capable of detection; and
also providing deoxyribonucleotide triphosphates of E'"'"' and F, a polymerase reagent and a ligase reagent;
c) performing the following cycle at least once;
i) mixing said probes with said sample under hybridizing conditions to allow probes to hybridize to the target sequence and its complement if present, or to reorganized probes created therefrom;
ii) using target sequence or reorganized probes created therefrom as template, extending probe A with said polymerase reagent by adding F deoxyribonucleotide triphosphates to its 3'"'"' end, and extending probe B'"'"' with said polymerase reagent by adding E'"'"' deoxyribonucleotide triphosphates to its 3'"'"' endiii) ligating extended probe A to probe B, and extended probe B'"'"' to probe A'"'"', using said ligase reagent to form reorganized probe molecules; and
iv) providing denaturing conditions to separate said reorganized probe molecules from said template;
d) separating reorganized probe molecules from unreorganized labeled probes; and
e) detecting the presence of said labeled probes in the reorganized or unreorganized fraction as a measure of the presence of the target sequence.
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Accused Products
Abstract
An improved, "gap filling" embodiment of the Ligase Chain Reaction (LCR) is described. Gap filling LCR is LCR wherein at least one of the probes is recessed so that a gap is formed between the adjacent probes when they are hybridized to target. The gap is filled using polymerase and deoxyribonucleotide triphosphates before ligation of the probes together. There are single and double gap versions, depending on whether one or two probes are recessed and require filling before ligation. The improvement resides in selecting and using target sequences such that only a single type, or two types, of deoxyribonucleotide triphosphate(s) are required to fill double gaps each being 1-10 bases in length, preferably 1-3 bases. Probes having specific sequences are claimed for a number of pathogens.
480 Citations
30 Claims
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1. A method for detecting the presence of a target nucleic acid in a sample, said method employing a ligase chain reaction to create geometrically increasing numbers of reorganized probe molecules in the presence of said target nucleic acid, said method comprising:
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a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula; 5'"'"'-(N)h XEp Fq Z(N)k -3'"'"' or its complement 3'"'"'-(N'"'"')h X'"'"'E'"'"'p F'"'"'q Z'"'"'(N'"'"')k -5'"'"', wherein E represents any base, E'"'"' is the complement of E, F represents any base except E, F'"'"' is the complement of F, p and q are independently integers from 1 to about 10, X represents any base except E or F'"'"', X'"'"' is the complement of X, Z represents any base except F or E'"'"', Z'"'"'is the complement of Z, N represents any base, and h and k are independently integers from 5 to 100, provided (p+h) and (q+k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected; b) providing a plurality of each of four probes having the formulas;
space="preserve" listing-type="equation">5'"'"'-(N).sub.h X E.sub.p -3'"'"' A
space="preserve" listing-type="equation">3'"'"'-(N'"'"').sub.h X'"'"'-5'"'"' A'"'"'
space="preserve" listing-type="equation">5'"'"'-Z(N).sub.k -3'"'"' B
space="preserve" listing-type="equation">3'"'"'-F'"'"'.sub.q Z'"'"'(N'"'"').sub.k -5'"'"'wherein N, N'"'"', X, X'"'"', Z, Z'"'"'E, F'"'"', h, p, k and q are defined above, except that A and A'"'"' or B and B'"'"' need not be identical in length, and wherein at least one of the probes is labeled with a moiety capable of detection; and
also providing deoxyribonucleotide triphosphates of E'"'"' and F, a polymerase reagent and a ligase reagent;c) performing the following cycle at least once; i) mixing said probes with said sample under hybridizing conditions to allow probes to hybridize to the target sequence and its complement if present, or to reorganized probes created therefrom; ii) using target sequence or reorganized probes created therefrom as template, extending probe A with said polymerase reagent by adding F deoxyribonucleotide triphosphates to its 3'"'"' end, and extending probe B'"'"' with said polymerase reagent by adding E'"'"' deoxyribonucleotide triphosphates to its 3'"'"' end iii) ligating extended probe A to probe B, and extended probe B'"'"' to probe A'"'"', using said ligase reagent to form reorganized probe molecules; and iv) providing denaturing conditions to separate said reorganized probe molecules from said template; d) separating reorganized probe molecules from unreorganized labeled probes; and e) detecting the presence of said labeled probes in the reorganized or unreorganized fraction as a measure of the presence of the target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method for detecting the presence of a target nucleic acid in a sample, said method employing a ligase chain reaction to create geometrically increasing numbers of reorganized probe molecules in the presence of said target nucleic acid, said method comprising:
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a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula; 5'"'"'-(N)h LEp E'"'"'q L(N)k -3'"'"' or its complement 3'"'"'-(N'"'"')h L'"'"'E'"'"'p Eq J'"'"'(N'"'"')k -5'"'"', wherein E represents any base, E'"'"' is the complement of E, p and q are independently integers from 1 to about 10, L represents any base except E, L'"'"' is the complement of L, J represents any base except E'"'"', J'"'"' is the complement of J, N represents any base, and h and k are independently integers from 5 to 100, provided (p+h) and (q+k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected; b) providing a plurality of each of four probes having the formulas;
space="preserve" listing-type="equation">5'"'"'-(N).sub.h L E.sub.p -3'"'"' A
space="preserve" listing-type="equation">3'"'"'-(N'"'"').sub.h L'"'"'-5'"'"' A'"'"'
space="preserve" listing-type="equation">5'"'"'-J(N).sub.k -3'"'"' B
space="preserve" listing-type="equation">3'"'"'-E'"'"'.sub.q J'"'"'(N'"'"').sub.k -5'"'"' B'"'"'wherein N, N'"'"', L, L'"'"', E, E'"'"', J, J'"'"'h, p, k and q are defined above, except that A and A'"'"' or B and B'"'"' need not be identical in length, and wherein at least one of the probes is labeled with a moiety capable of detection; and
also providing deoxyribonucleotide triphosphates of E'"'"', a polymerase reagent and a ligase reagent;c) performing the following cycle at least once; i) mixing said probes with said sample under hybridizing conditions to allow probes to hybridize to the target sequence and its complement if present; ii) using target sequence or reorganized probes created therefrom as template, extending probes A and B'"'"' with said polymerase reagent by adding E'"'"' deoxyribonucleotide triphosphates to their 3'"'"' ends iii) ligating extended probe A to probe B, and extended probe B'"'"' to probe A'"'"', using said ligase reagent to form reorganized probe molecules; and iv) providing denaturing conditions to separate said reorganized probe molecules from said template; d) separating reorganized probe molecules from unreorganized labeled probes; and e) detecting the presence of said labeled probes in the reorganized fraction as a measure of the presence of the target sequence. - View Dependent Claims (13, 14, 15, 16)
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17. A method for determining the presence of HIV 1 in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 1667, SEQ ID Nos 1 ,2 ,3 and 4; at map position 912, SEQ ID Nos. 5 ,6 ,7 and 8; at map position 2086, SEQ ID Nos. 9, 10, 11 and 12; at map position 789, SEQ ID Nos. 13, 14, 15 and 16; at map position 508, SEQ ID Nos. 17, 18, 19 and 20; at map position 5569, SEQ ID Nos. 21, 22, 23 and 24; at map position 1450, SEQ ID Nos. 25, 26, 27 and 28; at map position 1573, SEQ ID Nos. 29, 30, 31 and 32; at map position 4060, SEQ ID Nos. 33, 34, 35 and 36; at map position 2683, SEQ ID Nos. 37, 38, 39 and 40; at map position 1753, SEQ ID Nos. 41, 42, 43 and 44; and at map position 3697, SEQ ID Nos. 45, 46, 47 and 48.
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18. A method for determining the presence of HIV 2 in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 2060, SEQ ID Nos 49, 50, 51 and 52; at map position 4661, SEQ ID Nos. 53, 54, 55 and 56; and at map position 1094, SEQ ID Nos. 57, 58, 59 and 60.
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19. A method for determining the presence of HPV in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 631, SEQ ID Nos 61, 62, 63 and 64; at map position 480, SEQ ID Nos 65, 66, 67 and 68; at map position 488, SEQ ID Nos. 69, 70, 71 and 72; and at map position 6604, SEQ ID Nos. 73, 74, 75 and 76.
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20. A method for determining the presence of HSV in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 4751, SEQ ID Nos. 77, 78, 79 and 80; and at map position 6465, SEQ ID Nos. 81, 82, 83 and 84.
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21. A method for determining the presence of Chlamydia trachomatis in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 36, SEQ ID Nos 85, 86, 87 and 88; at map position 552, SEQ ID Nos. 89, 90, 91 and 92; and at map position 6693, SEQ ID Nos. 93, 94, 95 and 96.
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22. A method for determining the presence of Neisseria gonorrhoeae, in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 27, SEQ ID Nos 97, 98, 99 and 100; at map position 66, SEQ ID Nos 101, 102, 103 and 104; at map position 114, SEQ ID Nos 105, 106, 107 and 108; at map position 822, SEQ ID Nos. 109, 110, 111 and 112; and at map position 933, SEQ ID Nos. 113, 114, 115 and 116.
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23. A method for determining the presence of Borrelia burgdorferi in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe Sets consisting of:
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at map position 5, SEQ ID Nos. 117, 118, 119 and 120; and at map position 181, SEQ ID Nos. 121, 122, 123 and 124.
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24. A composition of matter useful for detecting HIV 1, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 1667, SEQ ID Nos 1, 2, 3 and 4; at map position 912, SEQ ID Nos. 5, 6, 7 and 8; at map position 2086, SEQ ID Nos. 9, 10, 11 and 12; at map position 789, SEQ ID Nos. 13, 14, 15 and 16; at map position 508, SEQ ID Nos. 17, 18, 19 and 20; at map position 5569, SEQ ID Nos. 21, 22, 23 and 24; at map position 1450, SEQ ID Nos. 25, 26, 27 and 28; at map position 1573, SEQ ID Nos. 29, 30, 31 and 32; at map position 4060, SEQ ID Nos. 33, 34, 35 and 36; at map position 2683, SEQ ID Nos. 37, 38, 39 and 40; at map position 1753, SEQ ID Nos. 41, 42, 43 and 44; and at map position 3697, SEQ ID Nos. 45, 46, 47 and 48.
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25. A composition of matter useful for detecting HIV 2, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe set consisting of:
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at map position 2060, SEQ ID Nos 49, 50, 51 and 52; at map position 4661, SEQ ID Nos. 53, 54, 55 and 56; and at map position 1094, SEQ ID Nos. 57, 58, 59 and 60.
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26. A composition of matter useful for detecting HPV, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe set consisting of:
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at map position 631, SEQ ID Nos 61, 62, 63 and 64; at map position 480, SEQ ID Nos 65, 66, 67 and 68; at map position 488, SEQ ID Nos. 69, 70, 71 and 72; and at map position 6604, SEQ ID Nos. 73, 74, 75 and 76.
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27. A composition of matter useful for detecting HSV, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 4751, SEQ ID Nos. 77, 78, 79 and 80; and at map position 6465, SEQ ID Nos. 81, 82, 83 and 84.
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28. A composition of matter useful for detecting Chlamydia trachomatis, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 36, SEQ ID Nos 85, 86, 87 and 88; at map position 552, SEQ ID Nos. 89, 90, 91 and 92; and at map position 6693, SEQ ID Nos. 93, 94, 95 and 96.
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29. A composition of matter useful for detecting Neisseria gonorrhoeae, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 27, SEQ ID Nos 97, 98, 99 and 100; at map position 66, SEQ ID Nos 101, 102, 103 and 104; at map position 114, SEQ ID Nos 105, 106, 107 and 108; at map position 822, SEQ ID Nos. 109, 110, 111 and 112; and at map position 933, SEQ ID Nos. 113, 114, 115 and 116.
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30. A composition of matter useful for detecting Borrelia burgdorferi, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:
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at map position 5, SEQ ID Nos. 117, 118, 119 and 120; and at map position 181, SEQ ID Nos. 121, 122, 123 and 124.
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Specification