Isothermal amplification of nucleic acids
First Claim
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1. An isothermal process for amplifying a single stranded nucleic acid template which comprises:
- (a) applying a forward primer (primer-1) to the 3′
region of the template;
(b) extending said primer by a polymerase;
(c) removing or degrading the 5′
terminus of said primer to leave a partly degraded duplex product (product-1);
(d) applying a further primer-1 molecule (primer-1a) to the region exposed by degradation of the 5′
terminus of the extended primer (primer-1) so that the 5′
region of said further primer (primer-1a) binds to the template;
(e) allowing strand invasion of product-1 by the 3′
terminus of said further primer (primer-1a), whereby the 3′
terminus of said further primer hybridises with the template in place of the 5′
terminus of product-1;
(f) extending said primer-1a by a strand displacing polymerase causing release of product-1 (amplicon) from the template; and
(g) reacting product-1 with a reverse primer (primer-2) which binds to the 3′
region of product-1;
(h) repeating steps (b) to (g) where the forward primer (primer-1) is replaced by the reverse primer (primer-2) in order to produce product-2 (amplicon);
wherein the original template is re-created by the reaction between product-2 and the 3′
terminus of primer-1, whereby the 5′
terminus of the primer is extended onto by the 3′
terminus of the template.
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Abstract
A process of amplifying a nucleic acid template dependent on partial destruction of primer molecules which have extended onto the template molecule followed by strand invasion of the partially destroyed primer template by a replacement primer. The destruction of the primer molecule may be performed by either endonuclease or exonuclease digestion. A signal generation from the amplified products may be obtained by the use of adaptors capable of binding probe molecules as well as the amplified product.
121 Citations
17 Claims
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1. An isothermal process for amplifying a single stranded nucleic acid template which comprises:
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(a) applying a forward primer (primer-1) to the 3′
region of the template;(b) extending said primer by a polymerase; (c) removing or degrading the 5′
terminus of said primer to leave a partly degraded duplex product (product-1);(d) applying a further primer-1 molecule (primer-1a) to the region exposed by degradation of the 5′
terminus of the extended primer (primer-1) so that the 5′
region of said further primer (primer-1a) binds to the template;(e) allowing strand invasion of product-1 by the 3′
terminus of said further primer (primer-1a), whereby the 3′
terminus of said further primer hybridises with the template in place of the 5′
terminus of product-1;(f) extending said primer-1a by a strand displacing polymerase causing release of product-1 (amplicon) from the template; and (g) reacting product-1 with a reverse primer (primer-2) which binds to the 3′
region of product-1;(h) repeating steps (b) to (g) where the forward primer (primer-1) is replaced by the reverse primer (primer-2) in order to produce product-2 (amplicon); wherein the original template is re-created by the reaction between product-2 and the 3′
terminus of primer-1, whereby the 5′
terminus of the primer is extended onto by the 3′
terminus of the template.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification