Polynomial amplification of nucleic acids
View Patent ↗The invention provides methods and compositions for the amplification and replication of nucleic acid molecules. In particular, novel amplification methods, referred to herein as polynomial amplification, are provided. According to these methods, a nucleic acid molecule to be amplified is contacted with at least two primers; a non-replicable primer which may hybridize to the nucleic acid molecule being amplified, and a replicable primer which may hybridize to a primer extension product generated from extension of the non-replicable primer.
1. A method for amplifying a nucleotide sequence of interest from a nucleic acid, which method comprises:
(a) contacting the nucleic acid with a primer set, which primer set comprises (i) a first non-replicable primer that hybridizes to the nucleic acid and (ii) a replicable primer that hybridizes to a primer extension product generated by replication of the nucleic acid from the first non-replicable primer; under conditions such that a first generation primer extension product that is capable of hybridizing to the replicable primer is produced using the nucleic acid as a template and the first non-replicable primer as the primer;
(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 and the nucleic acid 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 for the replicable primer, and the nucleic acid as a template for the first non-replicable primer; and
(d) repeating steps (b) and (c) as many times as desired to amplify the nucleotide sequence;
wherein the primer extension products contain at least a portion of the sequence of the nucleotide sequence of interest or its complement;
wherein the primer set further comprises a second non-replicable primer which hybridizes to the nucleic acid and to the second generation extension product; and
wherein the second non-replicable primer has a lower concentration than the first non-replicable primer.
2. The method of claim 1 wherein the second non-replicable primer is reversibly unable to initiate primer extension.
3. The method of claim 2 wherein the second non-replicable primer possesses a photolabile-blocking group which renders the second non-replicable primer unable to initiate primer extension.
4. The method of claim 1 wherein the second non-replicable primer has a lower melting temperature than the first non-replicable primer.
5. The method according to claim 1 , wherein the primer set further comprises a non-replicable primer which hybridizes to the (i) nucleic acid, (ii) to the second generation extension product; and (iii) to a primer extension product generated by replication from the replicable primer and extension products of the second non-replicable primer.