Methods for detecting small RNA species
First Claim
1. A method for determining the presence of a small target ribonucleotide sequence in a sample, said method comprising(a) modifying ribonucleotide species contained in the sample by polyadenylating the 3′
- ends;
(b) converting the polyadenylated ribonucleotide species into a plurality of complementary DNA (cDNA) sequences;
(c) immobilizing said plurality of cDNA sequences to a solid support;
(d) contacting said immobilized cDNA species with a pool of probe nucleic acids under conditions that allow sequence specific annealing, wherein each probe nucleic acid corresponds to a small target ribonucleotide sequence;
(e) extending said probe nucleic acids in a manner complementary to the immobilized cDNA species;
(f) removing the extended probe nucleic acids from the immobilized cDNA species;
(g) amplifying the extended probe nucleic acids to generate amplicons, and(h) detecting said amplicons, wherein detection of each amplicon indicates the presence of a small target ribonucleotide sequence.
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Abstract
The invention provides a method of detecting small target nucleotide sequences, in particular, small RNA species that are present in a sample. The method generally comprises a poly-A polymerization step or a ligation step to add a universal sequence to the 3′-end of all RNA molecules, followed by a universal primer-mediated cDNA synthesis, solid-phase selection, assay oligo annealing, extension and PCR amplification/labeling. The method of the invention can be practiced to amplify and label a small amount of miRNA or other ncRNA. The resulting amplification product can be read out on a universal array or an array with miRNA-specific or ncRNA-specific probes. The invention has multiple embodiments, including methods, compositions, and kits. In general, the nucleic acids, compositions, and kits comprise materials that are useful in carrying out the methods of the invention or are produced by the methods, and that can be used to detect small target nucleic acid sequences present in samples, in particular, small RNA species.
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Citations
16 Claims
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1. A method for determining the presence of a small target ribonucleotide sequence in a sample, said method comprising
(a) modifying ribonucleotide species contained in the sample by polyadenylating the 3′ - ends;
(b) converting the polyadenylated ribonucleotide species into a plurality of complementary DNA (cDNA) sequences; (c) immobilizing said plurality of cDNA sequences to a solid support; (d) contacting said immobilized cDNA species with a pool of probe nucleic acids under conditions that allow sequence specific annealing, wherein each probe nucleic acid corresponds to a small target ribonucleotide sequence; (e) extending said probe nucleic acids in a manner complementary to the immobilized cDNA species; (f) removing the extended probe nucleic acids from the immobilized cDNA species; (g) amplifying the extended probe nucleic acids to generate amplicons, and (h) detecting said amplicons, wherein detection of each amplicon indicates the presence of a small target ribonucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for determining the presence of a small target ribonucleotide sequence in a sample, said method comprising
(a) modifying ribonucleotide species in the sample by adding a chimera nucleic acid to the 3′ - end of the ribonucleotide species, wherein said chimera nucleic acid comprises RNA bases at its 5′
end and DNA bases at its 3′
end;(b) converting the modified ribonucleotide species into a plurality of complementary DNA (cDNA) sequences; (c) immobilizing said plurality of cDNA sequences to a solid support; (d) contacting said immobilized cDNA species with a pool of probe nucleic acids under conditions that allow sequence specific annealing, wherein each probe nucleic acid corresponds to a small target ribonucleotide sequence; (e) extending said probe nucleic acids in a manner complementary to the immobilized cDNA species; (f) removing the extended probe nucleic acids from the immobilized cDNA species; (g) amplifying the extended probe nucleic acids to generate amplicons, and (h) detecting said amplicons, wherein detection of each amplicon indicates the presence of a small target ribonucleotide sequence
- end of the ribonucleotide species, wherein said chimera nucleic acid comprises RNA bases at its 5′
Specification