Composition kits and methods for performing amplification reactions
First Claim
1. A kit for use in synthesizing multiple copies of an RNA target sequence contained within a target nucleic acid, the kit comprising:
- a priming oligonucleotide which hybridizes to the 3′
-end of the RNA target sequence and primes the synthesis of a first DNA primer extension product complementary to the RNA target sequence;
a cap hybridized to the 3′
-end of the priming oligonucleotide, the cap comprising a base region which is complementary to at least 3 nucleotides at the 3′
-end of the priming oligonucleotide, wherein the 5′
-terminal base of the cap is complementary to the 3′
-terminal base of the priming oligonucleotide, and wherein the cap cannot be extended by a nucleic acid polyerase;
a promoter oligonucleotide comprising a first region which hybridizes to a 3′
-region of the first DNA primer extension product and a second region which is a promoter for an RNA polymerase, the promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom; and
a binding molecule which binds to the target nucleic acid adjacent to or near the 5′
-end of the RNA target sequence,provided that any oligonucleotide included in the kit which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom.
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Accused Products
Abstract
The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side products. The method uses only one primer, the “priming oligonucleotide,” a 3′blocked promoter oligonucleotide and optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or eliminating the formation of side products. The method of the present invention minimizes or eliminates the emergence of side products, thus providing a high level of specificity. Furthermore, the appearance of side products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or eliminates this problem, thus providing an enhanced level of sensitivity.
38 Citations
86 Claims
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1. A kit for use in synthesizing multiple copies of an RNA target sequence contained within a target nucleic acid, the kit comprising:
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a priming oligonucleotide which hybridizes to the 3′
-end of the RNA target sequence and primes the synthesis of a first DNA primer extension product complementary to the RNA target sequence;a cap hybridized to the 3′
-end of the priming oligonucleotide, the cap comprising a base region which is complementary to at least 3 nucleotides at the 3′
-end of the priming oligonucleotide, wherein the 5′
-terminal base of the cap is complementary to the 3′
-terminal base of the priming oligonucleotide, and wherein the cap cannot be extended by a nucleic acid polyerase;a promoter oligonucleotide comprising a first region which hybridizes to a 3′
-region of the first DNA primer extension product and a second region which is a promoter for an RNA polymerase, the promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom; anda binding molecule which binds to the target nucleic acid adjacent to or near the 5′
-end of the RNA target sequence,provided that any oligonucleotide included in the kit which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A kit for use in synthesizing multiple copies of a DNA target sequence contained within a target nucleic acid, the kit comprising:
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a promoter oligonucleotide comprising a first region which hybridizes to a 3′
-region of the DNA target sequence present in a target nucleic acid to form a promoter oligonucleotide;
target nucleic acid hybrid and a second region which is a promoter for an RNA polymerase, the promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom;a priming oligonucleotide which hybridizes to the 3 ′
-end of an RNA product transcribed from the promoter oligonucleotide;
target nucleic acid hybrid and primes the synthesis of a first DNA primer extension product complementary to the RNA product, wherein the RNA product comprises a base region complementary to the DNA target sequence, and wherein the priming oligonucleotide does not comprise RNA,provided that the kit does not include a priming oligonucleotide which hybridizes to the target nucleic acid and primes the synthesis of a primer extension product complementary to the DNA target sequence, and further provided that the kit does not include a restriction endonuclease capable of cleaving a double-stranded complex comprising the target nucleic acid. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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53. A kit for use in synthesizing multiple copies of an RNA target sequence, the kit comprising:
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a priming oligonucleotide which hybridizes to the 3′
-end of the RNA target sequence and primes the synthesis of a first DNA primer extension product complementary to the RNA target sequence; anda cap which hybridizes to a region at the 3′
-end of the priming oligonucleotide, wherein the 5′
-terminal base of the cap is complementary to the 3′
-terminal base of the priming oligonucleotide, and wherein the cap is modified to prevent the initiation of DNA synthesis therefrom. - View Dependent Claims (54, 55, 56, 57, 58, 59, 60)
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61. A composition comprising:
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a promoter oligonucleotide having first and second regions, the first region comprising a 3 40 -portion containing a contiguous arrangement of ribonucleotides and a portion 5′
thereto that is capable of hybridizing to a DNA template, and the second region being a promoter for an RNA polymerase, wherein the first region is situated 3′
to the second region, and wherein the promoter oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom; andan oligodeoxynucleotide hybridized to the 3′
-portion of the first region, thereby forming an RNA;
DNA duplex. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78)
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79. A kit for use in synthesizing multiple copies of a DNA target sequence contained within a target nucleic acid, the kit comprising:
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a promoter oligonucleotide comprising a first region which hybridizes to a 3′
-region of the DNA target sequence present in a target nucleic acid to form a promoter oligonucleotide;
target nucleic acid hybrid and a second region which is a promoter for an RNA polymerase, the promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom;a priming oligonucleotide which hybridizes to the 3 ′
-end of an RNA product transcribed from the promoter oligonucleotide;
target nucleic acid hybrid and primes the synthesis of a first DNA primer extension product complementary to the RNA product, wherein the RNA product comprises a base region complementary to the DNA target sequence; anda cap hybridized to the 3′
-end of the priming oligonucleotide, the cap comprising a base region which is complementary to at least 3 nucleotides at the 3′
-end of the priming oligonucleotide, wherein the 5′
-terminal base of the cap is complementary to the 3′
-terminal base of the priming oligonucleotide, and wherein the cap cannot be extended by a nucleic acid polymerase,provided that the kit does not include a priming oligonucleotide which hybridizes to the target nucleic acid and primes the synthesis of a primer extension product complementary to the DNA target sequence, and further provided that the kit does not include a restriction endonuclease capable of cleaving a double-stranded complex comprising the target nucleic acid. - View Dependent Claims (80, 81, 82, 83, 84, 85, 86)
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Specification