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Highly selective nucleic acid amplification primers

  • US 9,909,159 B2
  • Filed: 02/07/2014
  • Issued: 03/06/2018
  • Est. Priority Date: 02/07/2013
  • Status: Active Grant
First Claim
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1. An amplification and detection method that is capable of detecting as few as ten copies of at least one rare mutant DNA target sequence in a mixture containing, for each mutant target sequence, 100,000 copies of a closely related wild-type DNA target sequence, comprising:

  • (a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a multi-part primer comprising, in the 5′

    to 3′

    direction the following three contiguous DNA sequences;

    an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing;

    a bridge sequence at least six nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing; and

    a foot sequence that is 5-8 nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by one or two nucleotides, wherein;

    (i) if the anchor sequence and the foot sequence of the primer are hybridized either to the mutant target sequence or to its closely related wild-type target sequence, there is in the target sequence an intervening sequence at least eight nucleotides long that does not hybridize to the primer'"'"'s bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of 28-52 nucleotides,(ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot/mutant-target-sequence hybrid that makes copying the intended target sequence unlikely as evidenced by a delay of at least five cycles in the threshold cycle (CT) of amplification of said at least one mutant target sequence using said multi-part primer as compared to using a conventional primer,(iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and(iv) the probability that during said cycling a multi-part primer/wild-type-target-sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a multi-part primer/mutant-target-sequence hybrid will be extended, as evidenced by a Δ

    CT of at least 13.3 cycles; and

    (b) detecting amplified product or products with a dsDNA binding dye, or for each multi-part primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each multi-part primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer'"'"'s 5′

    end that fluoresces only when incorporated in or hybridized to the primer'"'"'s amplified product.

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