Linked linear amplification of nucleic acids
First Claim
1. A process for amplifying a nucleic acid sequence of interest contained within a nucleic acid comprised of a first strand and a second strand that is complementary to the first strand, the process comprising:
- (a) contacting each of the first and second strands with a primer that contains a non-replicable element and/or a cleavable element, under conditions such that first generation primer extension products are produced using said strands as templates, and wherein the primer for the first strand is selected such that a first generation primer extension product produced using the first strand as a template, when separated from the first strand, can serve as a template for synthesis of a second generation primer extension product of the primer for the second strand;
(b) separating the first generation primer extension products from their respective templates to produce single-stranded molecules; and
(c) treating the first generation primer extension products with the primers of step (a) under conditions such that second generation primer extension products are produced using the first generation primer extension products as templates;
wherein the second generation primer extension products contain at least a portion of the sequence of the nucleic acid sequence of interest and no more than an insufficient portion of the binding site for the primer producing said first generation primer extension products.
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Abstract
The extensive synthesis ("amplification") of a nucleic acid sequence of interest is attained through a linked series of multi-cycle primer extension reactions (LLA). The primers used in each of the primer extension reactions of the process contain non-replicable elements that halt nucleic acid synthesis and thereby prevent the synthesized molecules from serving as templates in subsequent cycles. Synthesized molecules accumulate during primer extension in a mathematically linear fashion, thereby rendering the process relatively insensitive to contaminating nucleic acids. Multiple primer sets are employed, thereby ensuring the accumulation of a large number of copies of the nucleic acid sequence of interest. The invention also provides for the detection of an amplified nucleic acid sequence of interest, as well as reagent kits for carrying out the reaction.
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Citations
45 Claims
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1. A process for amplifying a nucleic acid sequence of interest contained within a nucleic acid comprised of a first strand and a second strand that is complementary to the first strand, the process comprising:
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(a) contacting each of the first and second strands with a primer that contains a non-replicable element and/or a cleavable element, under conditions such that first generation primer extension products are produced using said strands as templates, and wherein the primer for the first strand is selected such that a first generation primer extension product produced using the first strand as a template, when separated from the first strand, can serve as a template for synthesis of a second generation primer extension product of the primer for the second strand; (b) separating the first generation primer extension products from their respective templates to produce single-stranded molecules; and (c) treating the first generation primer extension products with the primers of step (a) under conditions such that second generation primer extension products are produced using the first generation primer extension products as templates; wherein the second generation primer extension products contain at least a portion of the sequence of the nucleic acid sequence of interest and no more than an insufficient portion of the binding site for the primer producing said first generation primer extension products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 32, 33, 34, 37, 38, 39)
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11. A process for amplifying a nucleic acid sequence of interest contained within complementary nucleic acid strands, comprising:
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(a) contacting the strands with first primers and second primers, each of said first and second primers containing a non-replicable element and/or a cleavable element, under conditions such that a first generation primer extension product is synthesized from said first and second primers using the strands as templates, and wherein the first and second primers are selected such that the first generation primer extension products, when separated from their templates, can serve as templates for synthesis of second generation primer extension products of the first and second primers; (b) separating the first generation primer extension products from their respective templates to produce single-stranded molecules; and (c) treating the first generation primer extension products with the first and second primers under conditions such that second generation primer extension products are synthesized using the first generation primer extension products as templates; wherein the second generation primer extension products contain at least a portion of the sequence of the nucleic acid sequence of interest and no more than an insufficient portion of the binding site for either of said first and second primers for producing said first generation primer extension products. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 40, 41, 42)
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22. A process for amplifying a nucleic acid sequence of interest contained within complementary first and second nucleic acid strands, comprising:
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(a) contacting the first and second strands with first primers and second primers, each of said first and second primers containing a non-replicable element and/or a non-cleavable element, under a first set of primer extension reaction conditions such that first generation primer extension products are produced from the first primers, and not from the second primers, using the first and second strands as templates, and wherein the first primers are selected such that a first generation primer extension product from this step, when separated from its template, can serve as a template for synthesis of a second generation extension product of the first primer for the complement of said strand; (b) separating the first generation primer extension products from their respective templates to produce single-stranded molecules; (c) treating the first generation primer extension products with the primers of step (a) under conditions such that second generation primer extension products are generated using first generation primer extension products as templates; wherein the second generation primer extension products contain at least a portion of the sequence of the nucleic acid sequence of interest and no more than an insufficient portion of the binding site for either of said first and second primers for producing said first generation primer extension products (d) separating the second generation primer extension products from their templates to produce single-stranded molecules; (e) repeating steps (c) and (d) at least once; and (f) subjecting the reaction mixture of step (e) to a second set of primer extension reaction conditions such that primer extension products are synthesized from said second primers using second generation primer extension products as templates. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 36, 43, 44, 45)
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Specification