Materials and methods for detection of nucleic acids
View Patent ↗Assays using non-natural bases are described. In one embodiment, the method involves contacting a sample suspected of containing the target nucleic acid with a polymerase and first and second primers; amplifying the target nucleic acid, if present in the sample, by PCR using the first and second primers to generate an amplification product having a double-stranded region and a single-stranded region that comprises the non-natural base; contacting the sample with a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the single-stranded region; annealing at least a portion of the reporter to the single-stranded region of the amplification product; and correlating a signal of the label with the presence of the target nucleic acid in the sample. The invention also provides corresponding kits for use in detecting target nucleic acids in a sample.
1. A method for detecting a target nucleic acid in a sample comprising:
a) amplifying the target nucleic acid using a reaction mixture comprising:
i. a pair of primers, wherein at least one primer of the pair comprises at least one non-natural base; and
ii. a nucleotide or oligonucleotide that comprises a non-natural base that base-pairs with the non-natural base of the at least one primer; and
b) detecting the target nucleic acid.
2. The method of claim 1 , wherein the target nucleic acid is detected during amplification.
3. The method of claim 1 , wherein the non-natural base is selected from the group consisting of iso-cytosine and iso-guanine.
4. The method of claim 1 , wherein the at least one primer further comprises a label.
5. The method of claim 4 , wherein the label is a fluorophore.
6. The method of claim 1 , wherein the nucleotide or oligonucleotide further comprises a label.
7. The method of claim 6 , wherein the label is a fluorophore.
8. The method of claim 6 , wherein the label is a quencher.
9. The method of claim 1 , wherein the at least one primer further comprises a first fluorophore and the nucleotide or oligonucleotide further comprises a second fluorophore, wherein the first fluorophore and second fluorophore exhibit fluorescence resonance energy transfer.
10. The method of claim 1 , wherein the at least one primer further comprises a fluorophore and the nucleotide or oligonucleotide further comprises a quencher that quenches the fluorophore.
11. The method of claim 1 , wherein the target nucleic acid is detected quantitatively.
12. The method of claim 1 , wherein the target nucleic acid comprises RNA and the method further comprises reverse transcribing the RNA.