Probes, libraries and kits for analysis of mixtures of nucleic acids and methods for constructing the same
The invention relates to nucleic acid probes, nucleic acid probe libraries, and kits for detecting, classifying, or quantitating components in a complex mixture of nucleic acids, such as a transcriptome, and methods of using the same. The invention also relates to methods of identifying nucleic acid probes useful in the probe libraries and to methods of identifying a means for detection of a given nucleic acid.
1. A library of oligonucleotide probes, wherein each probe in the library comprises a recognition sequence tag and a detection moiety, wherein the recognition sequence tag has at least one modified monomer analogue that increases the binding affinity for the complementary target sequence relative to the corresponding unmodified oligodeoxyribonucleotide,
wherein each probe has sufficient stability for sequence-specific binding and detection of its target sequence under stringent conditions,
wherein each probe has independently a total of 8 or 9 nucleotides,
wherein the library comprises a total of from 50 to 500 probes,
wherein the probes in the library hybridize to more than 90% of the mRNAs set forth in SEQ ID NOS: 47-35734, and
wherein at least one modified monomer analogue is a non-naturally occurring nucleotide analogue, a deoxyribose or ribose analogue, or an internucleotide linkage other than a phosphodiester linkage.
2. The library of claim 1 , wherein the detection moiety is a covalently or non-covalently bound minor groove binder or an intercalator selected from the group consisting of asymmetric cyanine dyes, DAPI, SYBR Green I, SYBR Green II, SYBR Gold, PicoGreen, thiazole orange, Hoechst 33342, Ethidium Bromide, 1-O-(1-pyrenylmethyl)glycerol, and Hoechst 33258.
3. The library of claim 1 , wherein the at least one modified monomer analogue in the recognition sequence tag comprises an LNA nucleotide.
4. The library of claim 1 , wherein at least one probe in the library comprises an SBC nucleobase, 2′-O-methyl, diamine purine, 2-thio uracil, 5-nitroindole, a universal base, intercalating nucleic acid, or minor-groove-binder.
5. The library of claim 1 , wherein at least one modified monomer analogue is the internucleotide linkage other than a phosphodiester linkage, and wherein the internucleotide linkage other than phosphodiester linkage is selected from the group consisting of alkyl phosphonate, phosphoramidite, alkyl-phosphotriester, phosphorothioate, and phosphorodithioate linkages.
6. The library of claim 4 , wherein at least one of the probes in the library comprises 5-nitroindole.
7. The library of claim 1 , wherein the detection moiety comprises a dual label that is detected by the 5′ nuclease assay principle.
8. The library of claim 1 , wherein at least one probe in the library has a DNA nucleotide at the 5′-end.
9. The library of claim 1 , wherein the detection moiety comprises an intercalating fluorophore or minor groove binder, the fluorescence of which is altered upon binding to a double-stranded DNA or DNA-RNA hetero-duplex.
10. The library of claim 1 , wherein each probe in the library targets more than 2,200 target sequences among SEQ ID NOS: 47-35734.
11. The library of claim 1 , wherein the detection moiety comprises a detectable label at:
(i) the 5′ or the 3′ terminus of the recognition sequence tag; or
(ii) the 5′ or the 3′ terminus of the probe.
12. The library of claim 11 , wherein the detection moiety comprises a detectable label at the 5′ or the 3′ terminus of the recognition sequence tag.
13. The library of claim 11 , wherein the detection moiety comprises detectable labels at the 5′ and 3′ termini of the recognition sequence tag, and the detectable labels are a fluorophore-quencher pair.
14. The library of claim 11 , wherein the detectable label at the 5′ or the 3′ terminus of the recognition sequence tag is a fluorescein.
15. The library of claim 7 , wherein at least one probe in the library further comprise a 5-nitroindole.
16. The library of claim 1 , wherein the recognition sequence tag in at least one probe in the library has a T m of at least 50° C., as measured in an aqueous solution of 10 mM Na 2 HPO 4 , 100 mM NaCl, and 0.1 mM EDTA, at pH 7.0.
17. The library of claim 1 , wherein the recognition sequence tag in at least one probe in the library has a T m of at least 60° C., as measured in an aqueous solution of 10 mM Na 2 HPO 4 , 100 mM NaCl, and 0.1 mM EDTA, at pH 7.0.