Solid support assay systems and methods utilizing non-standard bases
View Patent ↗Solid support assays using non-standard bases are described. A capture oligonucleotide comprising a molecular recognition sequence is attached to a solid support and hybridized with a target oligonucleotide. In some instances, the molecular recognition sequence includes one or more non-standard bases and hybridizes to a complementary tagging sequence of the target oligonucleotide. In other instances, incorporation of a non-standard base (e.g., via PCR or ligation) is used in the assay.
1. A method for assaying for one or more target oligonucleotides in a sample comprising:
(a) amplifying the one or more target oligonucleotides with a reaction mixture that comprises at least one amplification primer complementary to the one or more target oligonucleotides and forming an amplification product, wherein the at least one amplification primer comprises at least one non-standard base;
(b) contacting the amplification product of step (a) with at least one tagged primer that comprises a tagging sequence with at least one non-standard base and a sequence complementary to the amplification product;
(c) extending the at least one tagged primer and forming an extension product, wherein the extending step is carried out in the presence of a non-standard nucleotide triphosphate that is complementary to the at least one non-standard base of the at least one amplification primer;
(d) contacting the extension product with an oligonucleotide bound to a solid support, wherein the oligonucleotide comprises a molecular recognition sequence that is complementary to the tagging sequence; and
(e) detecting specific hybridization between the extension product and the oligonucleotide bound to the solid support.
2. The method of claim 1 , wherein the non-standard nucleotide triphosphate comprises a label or a coupling agent.
3. The method of claim 2 , wherein the coupling agent is biotin.
4. The method of claim 3 further comprising the step of coupling the biotin to a reporter.
5. The method of claim 4 , wherein the reporter is streptavidin-phycoerythrin.
6. The method of claim 1 , wherein the at least one amplification primer comprises a label.
7. The method of claim 1 , wherein the non-standard base is selected from the group consisting of iso-cytosine and iso-guanine.
8. The method of claim 1 , wherein the at least one tagged primer is an allele specific primer that comprises a nucleotide that is complementary to a polymorphic base of a target nucleic acid.
9. The method of claim 8 , wherein the nucleotide of the tagged primer that is complementary to the polymorphic base of the target nucleic acid is positioned within five bases from the 3′ end of the allele specific primer.
10. The method of claim 8 , wherein the allele specific primer has an allele specific tagging sequence.
11. The method of claim 1 , wherein the specific hybridization is detected without performing washing.
12. The method of claim 1 , wherein the specific hybridization is detected at room temperature.
13. The method of claim 1 , wherein the molecular recognition sequence that is complementary to the tagging sequence is 6 to 20 nucleotides.