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AssigneeDana-Farber Cancer Institute, Inc.
InventorGerassimos MAKRIGIORGOS
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This disclosure relates to compositions and methods for single-step, multi-stage amplification reactions that combine many stages of sample preparation process in a single tube reaction. The disclosed technology provides a mean of performing multiplexed nested PCR in a single vessel, without any need of purification steps, and is based on the use of three sets of primers: a pair of outer primers, a pair of inner primers that are nested within the pair of outer primers, and tail primers that are complementary to tails on the inner primers. By adjusting the temperature conditions, annealing temperatures of the primers, number of amplification cycles, and the concentrations of the outer, inner, and tail primers, it is possible to carry out multiplexed nested PCR in a single vessel.
A method of selecting and amplifying DNA targets in a single reaction vessel, the method comprising the following steps:
The method of any one of the preceding claims, wherein the annealing temperature of the tail primers is 3-20° C. different from the annealing temperature of the inner multiplexed primers.
The method of any one of the preceding claims, wherein the annealing temperature of the tail primers is 60-70° C.
The method of any one of the preceding claims, wherein the inner multiplexed primers are hot start primers, activated by subjecting the provided contents in the single reaction vessel to an activation temperature after the completion of step (b).
The method of any one of the preceding claims, wherein the tail primers are hot start primers, activated by subjecting the provided contents in the single reaction vessel to an activation temperature after the completion of step (c).
The method of any one of the preceding claims, wherein the concentration of outer multiplexed primers is 0.01-0.2 μM.
The method of any one of the preceding claims, wherein the concentration of inner multiplexed primers is 0.001-0.04 μM.
The method of any one of the preceding claims, wherein the concentration of tail primers is 0.1-1 μM.
The method of any one of the preceding claims, wherein the ratio of concentration of outer multiplexed primers to the concentration of inner multiplexed primers is 0.25-2000.
The method of any one of the preceding claims, wherein the ratio of the concentration of the tail primers to the concentration of inner multiplexed primers is 5-200.
The method of any one of the preceding claims, wherein the ratio of the concentration of the tail primer to the concentration of the outer multiplexed primers is 1-20.
The method of any one of the preceding claims, wherein the amplification of step (b) is carried out for 8-12 cycles.
The method of any one of the preceding claims, wherein the amplification of step (c) is carried out for 2-6 cycles.
The method of any one of the preceding claims, wherein the amplification of step (d) is carried out for 10-30 cycles.
The method of any one of the preceding claims, wherein the number of amplification cycles in step (b) exceeds the number of amplification cycles in step (c).
The method of any one of the preceding claims, wherein the number of amplification cycles in step (d) exceeds the number of amplification cycles in step (c).
The method of any one of the preceding claims, wherein the number of amplification cycles in step (d) exceeds the number of amplification cycles in step (b).
The method of any one of the preceding claims, wherein the tail primers further comprise 20-30 bp of a 3′end portion of a sequencing adapter.
The method of any one of the preceding claims, wherein the inner forward and inner reverse primers each further comprise, between the target-specific anchor and the common forward or reverse tails, a central portion that is a unique barcode.
The method of any one of the preceding claims, further comprising enriching mutant alleles of the target nucleic acids relative to wild-type alleles of the target nucleic acids after the completion of step (d).
The method of any one of the preceding claims, wherein step (d) comprises one or more of the following:
The method of claim 33, wherein the COLD-PCR, ice-COLD-PCR, TT-ice-COLD-PCR, or toehold PCR comprise the last amplification cycles of step (d).
The method of claim 34, wherein at least the first four amplification cycles of step (d) do not comprise COLD-PCR, ice-COLD-PCR, TT-ice-COLD-PCR, or toehold PCR.
The method of any one of the preceding claims, wherein DNA is obtained from a biological sample.
A reaction mixture comprising
A reaction mixture comprising
Dependencies are inferred from the claim text.