| CPC C12Q 1/6876 (2013.01) [C12Q 1/6832 (2013.01)] | 16 Claims |
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1. A DNA Detection Switch (DDS) probe system for concurrent amplification and selective detection of a target nucleotide sequence of interest in a sample comprising:
a first probe:antiprobe detection system for a liquid-phase hybridization comprising:
a) a probe oligonucleotide comprising:
a nucleotide sequence consisting of 14 bases to 24 bases that is complementary to a first target nucleotide sequence and a second target nucleotide sequence, said first target nucleotide sequence and the second target nucleotide sequence differing by at least one mismatched base; and
a fluorescence emitter attached thereto at its 5′ end or at its 3′ end; and
b) an antiprobe oligonucleotide in an excess amount over the amount of the probe oligonucleotide, said antiprobe oligonucleotide comprising:
a fluorescence modulator attached to an end of the antiprobe oligonucleotide that is opposite the 5′ end or the 3′ end of the probe oligonucleotide to which the fluorescence emitter is attached;
a nucleotide sequence that has an equal length of the nucleotide sequence of the probe oligonucleotide and fully complementary to the nucleotide sequence of the probe oligonucleotide sequence except for at least one mismatched base that is selected from A, T, G and C and located in a non-terminal, non-central position of the antiprobe oligonucleotide in a duplex formed by the probe oligonucleotide and the antiprobe oligonucleotide and said fluorescence modulator of the antiprobe oligonucleotide diminishes a fluorescent signal of the fluorescence emitter of the probe oligonucleotide when the antiprobe oligonucleotide hybridizes to the probe oligonucleotide, a duplex formed by the probe oligonucleotide and the first target nucleotide sequence and the duplex formed by the probe oligonucleotide and the antiprobe oligonucleotide differ by at least 2 kcal/mol in Gibbs free energy (G) and at least 4° C. in melting temperature (Tm), and a duplex formed by the probe oligonucleotide and the second target nucleotide sequence, and the duplex formed by the probe oligonucleotide and the first target nucleotide sequence differ by at least 4 kcal/mol in G and at least 8° C. in Tm such that in a solution, an affinity of the probe oligonucleotide to the antiprobe oligonucleotide is higher than an affinity of the probe oligonucleotide to the second target nucleotide sequence, and, an affinity of the probe oligonucleotide to the first target nucleotide sequence is higher than the affinity of the probe oligonucleotide to the antiprobe oligonucleotide and under solution hybridization conditions,
1) when the first target nucleotide sequence is present in the sample, after adding the probe oligonucleotide and the antiprobe oligonucleotide into the sample, the probe oligonucleotide preferentially forms a first duplex with the first target nucleotide sequence in the solution and the probe oligonucleotide not duplexed with the first target nucleotide sequence forms a second duplex with the antiprobe oligonucleotide in the solution thereby generating:
a first fluorescent intensity from the first duplex, said first fluorescent intensity from the first duplex proportional to an amount of the first target nucleotide sequence in the sample; and
a baseline second fluorescent intensity from the second duplex, said baseline second fluorescent intensity of the second duplex diminished relative to the first fluorescent intensity from the first duplex due to an interaction between the fluorescence emitter and the fluorescence modulator in the second duplex; or
2) when the first target nucleotide sequence and the second target nucleotide sequence are absent in the sample, after adding the probe oligonucleotide and the antiprobe oligonucleotide into the sample, the probe oligonucleotide preferentially forms the second duplex with the antiprobe oligonucleotide in the solution, thereby generating the baseline second fluorescent intensity from the second duplex; or
3) when the first target nucleotide sequence is absent in the sample and the second target nucleotide sequence is present in the sample, after adding the probe oligonucleotide and the antiprobe oligonucleotide into the sample, the probe oligonucleotide preferentially forms the second duplex with the antiprobe oligonucleotide in the solution and does not form a third duplex with the second target nucleotide sequence in the solution, thereby generating the baseline second fluorescent intensity from the second duplex.
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