Real-time nucleic acid detection processes and compositions
First Claim
1. A composition of matter comprising at least two parts:
- a first part comprising at least one first nucleic acid primer or nucleic acid construct that comprises (i) at least one first energy transfer element; and
(ii) nucleic acid sequences which are complementary to sequences in at least a portion of a nucleic acid of interest;
a second part comprising at least one second nucleic acid primer or nucleic acid construct that comprises;
(i) at least one second energy transfer element; and
(ii) nucleic acid sequences which are identical to nucleic acid sequences in at least a portion of said nucleic acid of interest;
wherein said first nucleic acid primer or nucleic acid construct does not comprise said second energy transfer element, and wherein said second nucleic acid primer or nucleic acid construct does not comprise said first energy transfer element.
1 Assignment
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Accused Products
Abstract
This invention provides for compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time nucleic acid detection allows for the qualitative or quantitative detection or determination of single-stranded or double-stranded nucleic acids of interest in a sample. Other processes are provided by this invention including processes for removing a portion of a homopolymeric sequence, e.g., poly A sequence or tail, from an analyte or library of analytes. Compositions useful in carrying out such removal processes are also described and provided.
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Citations
543 Claims
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1. A composition of matter comprising at least two parts:
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a first part comprising at least one first nucleic acid primer or nucleic acid construct that comprises (i) at least one first energy transfer element; and
(ii) nucleic acid sequences which are complementary to sequences in at least a portion of a nucleic acid of interest;
a second part comprising at least one second nucleic acid primer or nucleic acid construct that comprises;
(i) at least one second energy transfer element; and
(ii) nucleic acid sequences which are identical to nucleic acid sequences in at least a portion of said nucleic acid of interest;
wherein said first nucleic acid primer or nucleic acid construct does not comprise said second energy transfer element, and wherein said second nucleic acid primer or nucleic acid construct does not comprise said first energy transfer element. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 16, 17, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543)
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12. A composition of matter comprising at least two parts:
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a first part comprising at least two segments, wherein said first segment comprises a first nucleic acid primer or nucleic acid construct and at least one first energy transfer element, and said second segment comprises primer-extended complementary to at least a portion of a nucleic acid of interest; and
a second part comprising at least two segments, wherein said first segment of said second part comprises a second nucleic acid primer or nucleic acid construct and at least one second energy transfer element; and
wherein said second segment of said second part comprises a primer-extended nucleic acid sequence which is identical to at least a portion of said nucleic acid of interest;
wherein said first nucleic acid primer or nucleic acid construct does not comprise said second energy transfer element, and wherein said second nucleic acid primer or nucleic acid construct does not comprise said first nucleic acid energy element. - View Dependent Claims (13, 14, 15, 18, 19, 20, 21, 22, 23, 24)
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- 25. A composition of matter comprising two nucleic acid strands, wherein the first of said two strands comprises at least one first energy transfer element, and the second of said two strands comprises at least one second energy transfer element and wherein said first strand or a portion thereof is hybridized to said second strand or a portion thereof, and wherein said first strand lacks said second energy transfer element and said second strand lacks said first energy transfer element.
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55. A composition of matter that comprises a nucleic acid strand, said strand comprising two segments, the first segment comprising a primer or a nucleic acid construct;
- and said second segment comprising a primer-extended sequence,
wherein said primer or nucleic acid construct comprises a first energy transfer element, and wherein said primer-extended sequence comprises a second energy transfer element, and wherein said primer-extended sequence comprises more than one nucleotide. - View Dependent Claims (56, 57, 58, 59, 60, 61, 62, 63, 64, 65)
- and said second segment comprising a primer-extended sequence,
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66. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) a nucleic acid primer or nucleic acid construct that comprises;
(A) a nucleic acid sequence complementary to at least a portion of said nucleic acid of interest and (B) a first energy transfer element;
(iii) labeled nucleotide or nucleotides comprising a second energy transfer element; and
(iv) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii) and (iv) above;
(c) contacting under hybridization conditions said nucleic acid primer in said composition (ii) with said nucleic acid of interest;
(d) extending said nucleic acid primer by more than one nucleotide, thereby incorporating said labeled nucleotide or nucleotides; and
(e) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element in said nucleic acid primer and said second energy transfer element in an incorporated nucleotide. - View Dependent Claims (67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80)
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81. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) a first nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;
(iii) a second nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence identical to at least a portion of said one strand;
(iv) labeled nucleotide or nucleotides comprising a first energy transfer element; and
(v) reagents for carrying out nucleic acid strand extension;
wherein (ii), (iii) or both (ii) and (iii) comprise a second energy transfer element;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) contacting under hybridization conditions said first nucleic acid primer in said composition (ii) with one strand of said nucleic acid of interest and contacting under hybridization conditions said second nucleic acid primer in said composition (iii) with the complementary strand of said nucleic acid of interest if present;
(d) extending said first nucleic acid primer and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if said complementary strand is present, thereby incorporating said labeled nucleotide or nucleotides;
(e) separating said first primer-extended nucleic acid sequence from said nucleic acid of interest and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid of interest if present;
(f) contacting under hybridization conditions said first nucleic acid primer in said composition (ii) with said nucleic acid of interest or said second primer-extended nucleic acid sequence from step (e), and contacting under hybridization conditions said second nucleic acid primer in said composition (iii) with said first primer-extended nucleic acid sequence from step (e); and
(g) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between a second energy transfer element in said first nucleic acid primer, said second nucleic acid primer, or both, and a first energy element in an incorporated nucleotide. - View Dependent Claims (82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100)
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101. A composition of matter that comprises a nucleic acid strand, said strand comprising two segments, the first segment comprising a primer or a nucleic acid construct;
- and the second segment comprising a primer-extended sequence,
wherein said primer-extended sequence comprises one or more first energy transfer elements and one or more second energy transfer elements. - View Dependent Claims (102, 103, 104, 105, 106, 107, 108, 109, 110)
- and the second segment comprising a primer-extended sequence,
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111. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) a nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence complementary to at least a portion of said nucleic acid of interest and (iii) one or more first nucleotides, said first nucleotides comprising a first energy transfer element, and one or more second nucleotides, said second nucleotides comprising a second energy transfer element;
(iv) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii) and (iv) above;
(c) contacting under hybridization conditions said nucleic acid primer in said composition (ii) with said nucleic acid of interest;
(d) extending said nucleic acid primer, thereby incorporating said first and second nucleotides; and
(e) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element. - View Dependent Claims (112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127)
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128. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) a first nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;
(iii) a second nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence identical to at least a portion of said one strand;
(iv) one or more first nucleotides, said first nucleotides comprising a first energy transfer element, and one or more second nucleotides, said second nucleotides comprising a second energy transfer element;
and (v) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) contacting under hybridization conditions said first nucleic acid primer in said composition (ii) with one strand of said nucleic acid of interest and contacting under hybridization conditions said second nucleic acid primer in said composition (iii) with the complementary strand of said nucleic acid of interest if present;
(d) extending said first nucleic acid primer and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if said complementary strand is present, thereby incorporating said first and second nucleotides; and
(e) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element. - View Dependent Claims (129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146)
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- 147. A composition of matter that comprises a nucleic acid strand, said strand comprising two or more ribonucleotides, wherein at least one ribonucleotide comprises a first energy transfer element and wherein at least one other ribonucleotide comprises a second energy transfer element.
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153. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) at least one first nucleic acid primer or nucleic acid construct and optionally, a second nucleic acid primer or nucleic acid construct, wherein either the first nucleic acid primer or nucleic acid construct, or said optional second nucleic acid primer or nucleic acid construct comprises an RNA promoter sequence;
(iii) one or more first ribonucleotides, said first ribonucleotides comprising a first energy transfer element, and one or more second ribonucleotides, said second ribonucleotides comprising a second energy transfer element;
(iv) reagents for carrying out nucleic acid strand extension and RNA transcription;
(b) forming a reaction mixture comprising (i), (ii) and (iii) above;
(c) contacting under hybridization conditions said first nucleic acid primer or nucleic acid construct (ii) with one strand of said nucleic acid of interest;
(d) extending said nucleic acid primer to form a primer-extended nucleic acid sequence;
(e) synthesizing a second nucleic acid strand complementary to said primer-extended nucleic acid sequence or a portion thereof, thereby forming a double-stranded nucleic acid;
(f) transcribing said double-stranded nucleic acid formed in step (e) to incorporate said first and second ribonucleotides (iii) into transcripts;
(g) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element;
wherein said transcribing step (f) is carried out by said RNA promoter sequence in either the primer-extended nucleic acid sequence or the second nucleic acid strand synthesized in step (e). - View Dependent Claims (154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168)
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169. A composition of matter that comprises two parts, wherein the first part is a nucleic acid strand comprising two segments, the first segment comprising a nucleic acid primer or nucleic acid construct, and the second segment comprises a primer-extended sequence, and
wherein the second part comprises a nucleic acid binding agent, wherein said nucleic acid primer or nucleic acid construct comprises one or more fluorescent first energy transfer elements and wherein said nucleic acid binding agent comprises one or more second energy transfer elements.
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180. A composition of matter that comprises
(a) two nucleic acid strands, wherein at least one of said strands comprises two segments, the first segment comprising a nucleic acid primer or nucleic acid construct, and the second segment comprising a primer-extended sequence, and (b) a nucleic acid binding agent, wherein said nucleic acid primer or nucleic acid construct comprises one or more fluorescent first energy transfer elements and wherein said nucleic acid binding agent comprises one or more second energy transfer elements.
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195. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) a nucleic acid primer or nucleic acid construct that comprises (A) a nucleic acid sequence complementary to at least a portion of said nucleic acid of interest; and
(B) one or more fluorescent first energy transfer elements;
(iii) a nucleic acid binding agent comprising one or more second energy transfer elements;
(iv) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii) and (iv) above;
(c) contacting under hybridization conditions said nucleic acid primer in said composition (ii) with said nucleic acid of interest;
(d) extending said nucleic acid primer or nucleic acid construct to form a primer-extended sequence;
(e) binding said nucleic acid binding agent (iii) to said primer-extended sequence or to a complex comprising said nucleic acid of interest and said primer-extended sequence; and
(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element. - View Dependent Claims (196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214)
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215. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) a first nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;
(iii) a second nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence identical to at least a portion of said one strand;
(iv) a nucleic acid binding agent comprising one or more first energy transfer elements; and
(v) reagents for carrying out nucleic acid strand extension;
wherein (ii), (iii) or both (ii) and (iii) comprise one or more fluorescent second energy transfer elements;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) contacting under hybridization conditions said first nucleic acid primer in said composition (ii) with one strand of said nucleic acid of interest and contacting under hybridization conditions said second nucleic acid primer in said composition (iii) with the complementary strand of said nucleic acid of interest if present;
(d) extending said first nucleic acid primer and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if the complementary strand is present;
(e) binding said nucleic acid binding agent (iv) to a primer-extended nucleic acid sequence or to a complex comprising said nucleic acid of interest and a primer-extended sequence; and
(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between a second energy transfer element in said first nucleic acid primer, said second nucleic acid primer, or both, and a first energy element in said nucleic acid binding agent (iv). - View Dependent Claims (216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 244)
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238. A composition of matter that comprises
(a) a nucleic acid strand comprising two segments, said first segment comprising a primer or a nucleic acid construct; - and said second segment comprising a primer-extended sequence; and
(b) a nucleic acid binding agent;
wherein said primer-extended sequence comprises one or more first energy transfer elements, and wherein said nucleic acid binding agent comprises one or more second energy transfer elements. - View Dependent Claims (239, 240, 241, 242, 243, 245, 246, 247, 248)
- and said second segment comprising a primer-extended sequence; and
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249. A composition of matter that comprises
(a) two nucleic acid strands, wherein at least one of said strands comprises two segments, the first segment comprising a nucleic acid primer or nucleic acid construct, and the second segment comprising a primer-extended sequence, and (b) a nucleic acid binding agent, wherein said primer-extended sequence comprises one or more first energy transfer elements, and wherein said nucleic acid binding agent comprises one or more second energy transfer elements.
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264. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) at least one nucleic acid primer or nucleic acid construct complementary to at least a portion of said nucleic acid of interest;
(iii) labeled nucleotide or nucleotides comprising a first energy transfer element;
(iv) a nucleic acid binding agent comprising one or more second energy transfer elements; and
(v) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) contacting under hybridization conditions said nucleic acid primer in said composition (ii) with said nucleic acid of interest;
(d) extending said nucleic acid primer, thereby incorporating said labeled nucleotide or nucleotides;
(e) binding said nucleic acid binding agent to said primer-extended sequence; and
(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element. - View Dependent Claims (265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284)
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285. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing;
(i) a sample suspected of containing said nucleic acid of interest;
(ii) a first nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;
(iii) a second nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence identical to at least a portion of said one strand;
(iv) one or more labeled nucleotides comprising a first energy transfer element;
(v) a nucleic acid binding agent comprising one or more second energy transfer elements; and
(vi) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv), (v) and (vi) above;
(c) contacting under hybridization conditions said first nucleic acid primer or nucleic acid construct (ii) with one strand of said nucleic acid of interest and contacting under hybridization conditions said second nucleic acid primer or nucleic acid construct (iii) with the complementary strand of said nucleic acid of interest if present;
(d) extending said first nucleic acid primer and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if the complementary strand is present, thereby incorporating said labeled nucleotide or nucleotides;
(e) binding said nucleic acid binding agent (v) to said first primer-extended nucleic acid sequence, and to said second primer-extended nucleic acid sequence if the complementary strand is present; and
(f) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element. - View Dependent Claims (286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306)
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307. A composition of matter that comprises:
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(i) a nucleic acid strand, said strand comprising one or more ribonucleotides, wherein at least one ribonucleotide comprises a first energy transfer element; and
(ii) a nucleic acid binding agent comprising one or more second energy transfer elements. - View Dependent Claims (308, 309, 310, 311, 312, 313, 314, 315, 316)
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317. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) at least one first nucleic acid primer or nucleic acid construct and optionally, a second nucleic acid primer or nucleic acid construct, wherein either the first nucleic acid primer or nucleic acid construct, or said optional second nucleic acid primer or nucleic acid construct comprises an RNA promoter sequence;
(iii) one or more labeled ribonucleotides comprising a first energy transfer element;
(iv) a nucleic acid binding agent comprising a second energy transfer element;
(v) reagents for carrying out nucleic acid strand extension and RNA transcription;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) contacting under hybridization conditions said first nucleic acid primer or nucleic acid construct (ii) with one strand of said nucleic acid of interest;
(d) extending said nucleic acid primer to form a primer-extended nucleic acid sequence;
(e) synthesizing a second nucleic acid strand complementary to said primer-extended nucleic acid sequence or a portion thereof, thereby forming a double-stranded nucleic acid;
(f) transcribing said double-stranded nucleic acid formed in step (e) to incorporate said labeled ribonucleotides (iii) into labeled transcripts;
(g) binding said nucleic acid binding agent (iv) to said labeled transcripts;
(h) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element;
wherein said transcribing step (f) is carried out by an RNA promoter in either the primer-extended nucleic acid sequence or the second nucleic acid strand synthesized in step (e). - View Dependent Claims (318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335)
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336. A composition of matter that comprises a hybridized first and second nucleic acids,
said first strand comprising two segments, the first segment comprising a primer or a nucleic acid construct; - and the second segment comprising a primer-extended sequence,
said second strand comprising a nucleic acid probe hybridized to said primer-extended sequence or a portion thereof, and wherein said primer-extended sequence comprises one or more first energy transfer elements, and wherein said nucleic acid probe comprises one or more second energy transfer elements. - View Dependent Claims (337, 338, 339, 340, 341, 342, 343)
- and the second segment comprising a primer-extended sequence,
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344. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) a nucleic acid primer or nucleic acid construct complementary to at least a portion of said nucleic acid of interest;
(iii) labeled nucleotide or nucleotides comprising a first energy transfer element;
(iv) a nucleic acid probe comprising one or more second energy transfer elements; and
(v) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) contacting under hybridization conditions said nucleic acid primer in said composition (ii) with said nucleic acid of interest;
(d) extending said nucleic acid primer, thereby incorporating said labeled nucleotide or nucleotides;
(e) separating said primer-extended sequence from said nucleic acid of interest (i);
(f) hybridizing said nucleic acid probe to said primer-extended sequence; and
(g) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element. - View Dependent Claims (345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359)
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360. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing;
(i) a sample suspected of containing said nucleic acid of interest;
(ii) a first nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;
(iii) a second nucleic acid primer or nucleic acid construct that comprises a nucleic acid sequence identical to at least a portion of said one strand;
(iv) one or more labeled nucleotides comprising a first energy transfer element;
(v) a nucleic acid probe comprising one or more second energy transfer elements; and
(vi) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv), (v) and (vi) above;
(c) contacting under hybridization conditions said first nucleic acid primer or nucleic acid construct (ii) with one strand of said nucleic acid of interest and contacting under hybridization conditions said second nucleic acid primer or nucleic acid construct (iii) with the complementary strand of said nucleic acid of interest if present;
(d) extending said first nucleic acid primer and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if the complementary strand is present, thereby incorporating said labeled nucleotide or nucleotides;
(e) separating said first primer-extended nucleic acid sequence and said second primer-extended nucleic acid sequence if produced in step (d);
(f) hybridizing said nucleic acid probe (v) to said first primer-extended nucleic acid sequence, or to said second primer-extended nucleic acid sequence; and
(g) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element. - View Dependent Claims (361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377)
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378. A composition of matter that comprises a hybridized first and second nucleic acids, said first strand comprising at least one ribonucleotide, wherein said at least one ribonucleotide comprises a first energy transfer element;
said second strand comprising;
a nucleic acid probe hybridized to said first strand or a portion thereof, wherein said nucleic acid probe comprises one or more second energy transfer elements. - View Dependent Claims (379, 380, 381, 382, 383, 384, 385, 386)
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387. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid of interest;
(ii) at least one first nucleic acid primer or nucleic acid construct and optionally, a second nucleic acid primer or nucleic acid construct, wherein either the first nucleic acid primer or nucleic acid construct, or said optional second nucleic acid primer or nucleic acid construct comprises an RNA promoter sequence;
(iii) one or more labeled ribonucleotides comprising a first energy transfer element;
(iv) a nucleic acid binding probe comprising a second energy transfer element;
(v) reagents for carrying out nucleic acid strand extension and RNA transcription;
(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) contacting under hybridization conditions said first nucleic acid primer or nucleic acid construct (ii) with one strand of said nucleic acid of interest;
(d) extending said nucleic acid primer to form a primer-extended nucleic acid sequence;
(e) synthesizing a second nucleic acid strand complementary to said primer-extended nucleic acid sequence or a portion thereof, thereby forming a double-stranded nucleic acid;
(f) transcribing said double-stranded nucleic acid formed in step (e) to incorporate said labeled ribonucleotides (iii) into labeled transcripts;
(g) hybridizing said nucleic acid probe (iv) to said labeled transcripts;
(h) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element;
wherein said transcribing step (f) is carried out by an RNA promoter in either the primer-extended nucleic acid sequence or the second nucleic acid strand synthesized in step (e). - View Dependent Claims (388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404)
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- 405. A chimeric nucleic acid construct comprising at least two segments, the first segment comprising five or more universal bases and the second segment comprising a nucleic acid template for synthesizing a discrete nucleic acid sequence.
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414. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:
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a) providing (i) said analyte or library of analytes;
(ii) a chimeric nucleic acid construct comprising at least two segments, the first segment comprising five or more universal bases and the second segment comprising a template for synthesizing said desirable nucleic acid sequence; and
(iii) reagents for carrying out hybridization and template-dependent strand extension;
b) binding or hybridizing said chimeric nucleic acid construct (ii) to said analyte or library of analytes (i); and
c) extending the 3′
end of said analyte or library of analytes;
wherein said second segment is a template for said extending step (c) when said second segment is in proximity to the 3′
end of said analyte or library of analytes. - View Dependent Claims (415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435)
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436. A composition comprising a set of chimeric nucleic acid constructs, each such construct comprising at least three segments, the first segment comprising four or more universal bases, the second segment comprising permutations of from one to eight nucleotides, the third segment comprising a template for synthesizing a desirable nucleic acid sequence,
wherein in said second segment, the permutations of from one to eight nucleotides comprise
(N)nwherein N independently comprises G, A, T or C, and wherein n is an integer from one to eight.
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448. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:
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a) providing (i) said analyte or library of analytes;
(ii) a set of chimeric nucleic acid constructs, each such construct comprising at least three segments, the first segment comprising four or more universal bases, the second segment comprising permutations of from one to eight nucleotides, the third segment comprising a template for synthesizing said desirable nucleic acid sequence, wherein in said second segment, the permutations of from one to eight nucleotides comprise
(N)nwherein N independently comprises G, A, T or C, and wherein n is an integer from one to eight; and
(iii) reagents for carrying out hybridization and template-dependent strand extension;
b) binding or hybridizing said chimeric nucleic acid construct (ii) to said analyte or library of analytes (i); and
c) extending the 3′
end of said analyte or library of analytes;
wherein said third segment is a template for said extending step (c) when said second segment is hybridized to the 3′
end of said analyte or library of analytes. - View Dependent Claims (449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 464, 465, 466, 467, 468, 469, 470, 471)
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472. A chimeric nucleic acid construct that comprises at least two segments,
a first segment comprising four or more universal bases; - and
a second segment which is complementary to a discrete nucleic acid sequence of interest;
wherein the 5′
end of said first segment is attached to the 3′
end of said second segment, and the 5′
end of said second segment is attached to the 3′
end of said third segment. - View Dependent Claims (473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496)
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497. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:
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(a) providing;
(i) said analyte or library of analytes;
(ii) a chimeric nucleic acid construct comprising at least two segments, wherein the first of said two segments is complementary to a first analyte nucleic acid sequence and wherein the second of said two segments is complementary to a second analyte nucleic acid sequence;
(iii) reagents for hybridization and endonucleolytic digestion;
(iv) an endonuclease; and
(v) reagents for strand extension;
(b) forming a mixture comprising (i), (ii), (iii) and (iv);
(c) hybridizing said chimeric nucleic acid construct (ii) with said analyte or library of analytes (i), thereby forming a first complex between said first segment and said first analyte nucleic acid sequence, and a second complex between said second segment and said second analyte nucleic acid sequence;
wherein said first complex is resistant to digestion by said endonuclease, and wherein said second complex comprises one or more sites sensitive to endonucleolytic digestion;
(d) digesting said second complex with said endonuclease (iv); and
(e) extending the 3′
-end of said second analyte nucleic acid sequence, said 3′
-end having been generated by said endonuclease (iv),wherein said extending step (e) is carried out by template dependent polymerization or ligation. - View Dependent Claims (498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515)
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516. A chimeric nucleic acid construct that comprises at least two nucleic acid segments, wherein the first segment and the second segment each comprise sequences complementary to different portions of a homopolymeric sequence in a nucleic acid of interest,
wherein hybridization of said first segment to a first portion of said homopolymeric sequence produces a first complex; -
wherein hybridization of said second segment to a second portion of said homopolymeric sequence produces a second complex; and
wherein the second portion of the homopolymeric sequence in said second complex is a substrate for a specific endonuclease, and wherein said first complex is resistant to said specific endonuclease. - View Dependent Claims (517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527)
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Specification