Switch-like isothermal dna amplification demonstrating a non-linear amplification rate
OwnerStephanie E. McCalla, Tomas Gedeoninventors
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Abstract
The presently-disclosed subject matter generally relates to methods, systems, compositions, and kits for the rapid, isothermal amplification of nucleic acids. The chemistry of the presently-disclosed amplification technique is isothermal, can be adapted to respond to a broad range of input target molecules, and results in a novel, biphasic reporter oligonucleotide amplification scheme with a high-gain second phase “burst” demonstrating a non-linear amplification rate (i.e., cooperative Hill kinetics). The switch-like amplification technique acts decisively to a true signal while filtering out noise, thus eliminating high levels of non-specific background amplification and false-positives.
Claims28
A method of detecting a target oligonucleotide sequence (X), said method comprising:
- (A) forming a reaction mixture that comprises:
- (1) a target nucleic acid comprising a target oligonucleotide sequence (X);
- (2) a first antisense template (X′R1t′YP) that comprises from 3′ to 5′:
- (a) a first sequence of nucleotides (X′) that is at least substantially complementary to the target oligonucleotide sequence (X);
- (b) a second sequence of nucleotides (R1) that is an anti-sense strand of a first nicking enzyme binding site;
- (c) a third sequence of nucleotides (t′Yp) that is at least substantially complementary to a reporter oligonucleotide sequence (tYp), comprising from 3′ to 5′:
- (i) a toehold nucleotide sequence (t′);
- and
- (ii) a palindromic nucleotide sequence (Yp);
- (3) a second antisense template (t′YpR2t′Yp) that comprises from 3′ to 5′:
- (a) a fourth sequence of nucleotides t′Yp;
- (b) a fifth sequence of nucleotides (R2) that is an anti-sense strand of a second nicking enzyme binding site;
- (c) a sixth sequence of nucleotides t′Yp;
- wherein the two palindromic nucleotide sequences (Yp) cause the second antisense template (t′YpR2t′YP) to form a palindrome and fold into a stem and loop configuration;
- (4) a polymerase;
- (5) a first nicking enzyme that nicks at the first nicking enzyme binding site;
- (6) a second nicking enzyme that nicks at the second nicking enzyme binding site;
- (7) nucleotides;
- (B) subjecting the reaction mixture to essentially isothermal conditions at a reaction temperature to amplify the reporter oligonucleotide sequence (tYp) at a non-linear amplification rate;
- and
- (C) detecting the amplified reporter oligonucleotide sequence (tYp).
Dependencies are inferred from the claim text.