METHODS AND KITS FOR AMPLIFYING DNA
First Claim
1. A method of synthesizing multiple copies of a target sequence, said method comprising:
- treating a target nucleic acid comprising a DNA target sequence with a priming oligonucleotide which hybridizes to the 3′
-end of said target sequence such that a primer extension reaction can be initiated therefrom;
extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a first DNA primer extension product, at least a portion of said first primer extension product being complementary to said target sequence;
treating said first primer extension product with a promoter oligonucleotide comprising first and second regions, said first region comprising a base sequence which corresponds to a region at the 5′
-end of said target sequence and which hybridizes to said first primer extension product to form a promoter oligonucleotide;
first primer extension product hybrid, and said second region comprising a promoter for an RNA polymerase and situated 5′
to said first region, wherein said promoter oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom;
transcribing from said promoter oligonucleotide;
first primer extension product hybrid multiple first RNA products complementary to at least a portion of said first primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said first RNA products are substantially identical to the base sequence of said target sequence, provided that if said first primer extension product has a defined 3′
-end, then said method further comprises treating said target nucleic acid with a binding molecule which binds to said target nucleic acid adjacent to or near the 5′
-end of said target sequence, and further provided that said priming oligonucleotide does not include an RNA region which hybridizes to said target nucleic acid and which is selectively degraded by an enzyme activity when hybridized to said target nucleic acid.
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Abstract
Novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic are disclosed (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, methods of nucleic acid amplification are disclosed which are robust and efficient, while reducing the appearance of side-products. In general, the methods use priming oligonucleotides that target only one sense of a target nucleic acid, a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-pro ducts. The disclosed methods minimizes or substantially eliminate 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 disclosed methods minimize or substantially eliminate this problem, thus providing enhanced levels of sensitivity.
89 Citations
56 Claims
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1. A method of synthesizing multiple copies of a target sequence, said method comprising:
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treating a target nucleic acid comprising a DNA target sequence with a priming oligonucleotide which hybridizes to the 3′
-end of said target sequence such that a primer extension reaction can be initiated therefrom;
extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a first DNA primer extension product, at least a portion of said first primer extension product being complementary to said target sequence;
treating said first primer extension product with a promoter oligonucleotide comprising first and second regions, said first region comprising a base sequence which corresponds to a region at the 5′
-end of said target sequence and which hybridizes to said first primer extension product to form a promoter oligonucleotide;
first primer extension product hybrid, and said second region comprising a promoter for an RNA polymerase and situated 5′
to said first region, wherein said promoter oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom;
transcribing from said promoter oligonucleotide;
first primer extension product hybrid multiple first RNA products complementary to at least a portion of said first primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said first RNA products are substantially identical to the base sequence of said target sequence,provided that if said first primer extension product has a defined 3′
-end, then said method further comprises treating said target nucleic acid with a binding molecule which binds to said target nucleic acid adjacent to or near the 5′
-end of said target sequence, andfurther provided that said priming oligonucleotide does not include an RNA region which hybridizes to said target nucleic acid and which is selectively degraded by an enzyme activity when hybridized to said target nucleic acid. - 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, 31, 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 amplifying a DNA target sequence, said kit comprising:
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a priming oligonucleotide which hybridizes to the 3′
-end of a DNA target sequence such that a primer extension reaction can be initiated therefrom; and
a promoter oligonucleotide comprising first and second regions, wherein said first region has abase sequence which corresponds to a region at the 5′
-end of said target sequence and which hybridizes to a 3′
-region of a DNA primer extension product comprising said priming oligonucleotide, wherein said second region is a promoter for an RNA polymerase and is situated 5′
to said first region, and wherein said promoter oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom,provided that any oligonucleotide included in said kit that comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom, provided that said kit does not include a restriction endonuclease, and further provided that said priming oligonucleotide does not include an RNA region which hybridizes to said target nucleic acid and which is selectively degraded by an enzyme activity when hybridized to said target nucleic acid. - View Dependent Claims (54, 55, 56)
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Specification