Probes, libraries and kits for analysis of mixtures of nucleic acids and methods for constructing the same

A library of oligonucleotide probes according to any one of the preceding claims, wherein more than 90% of the oligonucleotide probes can bind and detect at least two target sequences in a nucleic acid population.

A library of oligonucleotide probes of any one of the preceding claims, wherein the number of different target sequences in a nucleic acid population is at least 100.

A library of oligonucleotide probes according to any one of the preceding claims, wherein at least one nucleotide in each oligonucleotide probe is substituted with a non-naturally occurring nucleotide analogue, a deoxyribose or ribose analogue, or an internucleotide linkage other than a phosphodiester linkage.

A library of oligonucleotide probes according to any one of the preceding claims, wherein the detection moiety is a covalently or non-covalently bound minor groove binder or an intercalator selected from the group comprising 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.

The library of oligonucleotide probes according to any one of the preceding claims, wherein said oligonucleotide probes contain non-naturally occurring nucleotides, such as 2′-O-methyl, diamine purine, 2-thio uracil, 5-nitroindole, universal or degenerate bases, intercalating nucleic acids or minor-groove-binders, to enhance their binding to a complementary nucleic acid sequence.

The library of oligonucleotide probes according to any one of the preceding claims, wherein said different recognition sequences comprise less than 1% of all possible oligonucleotides of a given length.

The library of oligonucleotide probes according to any one of the preceding claims, wherein each probe can be detected using a dual label by the molecular beacon assay principle.

The library according to any one of the preceding claims, wherein each probe contains a single detection moiety that can be detected by the molecular beacon assay principle.

The library of oligonucleotide probes according to any one of the preceding claims, wherein the target nucleic acid population is an mRNA sample, a cDNA sample or a genomic DNA sample.

The library of oligonucleotide probes according to any one of the preceding claims, wherein said probe target sequences occur at least once within more than 4% of different target nucleic acids in a target nucleic acid population.

The library of oligonucleotide probes according to any one of the preceding claims, wherein self-complementary probe sequences have been omitted from the said library.

The library of oligonucleotide probes according to any one of the preceding claims, wherein the melting temperature (Tm) of each probe is adjusted to be suitable for PCR-based assays by substitution with non-occurring modifications, such as LNA, optionally modified with SBC nucleobases, 2′-O-methyl, diamine purine, 2-thio uracil, 5-nitroindole, universal or degenerate bases, intercalating nucleic acids or minor-groove-binders, to enhance their binding to a complementary nucleic acid sequence.

The library of oligonucleotide probes according to any one of the preceding claims, wherein the melting temperature (Tm) of each probe is at least 50° C.

The library of oligonucleotide probes according to any one of the preceding claims, wherein each probe has a DNA nucleotide at the 5′-end.

The library of oligonucleotide probes according to any one of the preceding claims, wherein each probe contains a fluorophore-quencher pair for detection.

The library of oligonucleotide probes according to any one of the preceding claims, wherein each probe can be detected by the molecular beacon principle.

The library of oligonucleotide probes according to any one of the preceding claims, wherein each probe is attached to an intercalating fluorophore or minor groove binder, which upon binding to a double-stranded DNA or DNA-RNA hetero-duplex target alter the fluorescence.

The library of oligonucleotide probes according to any one of the preceding claims, wherein the target population is the human transcriptome.

The library of oligonucleotide probes according to any one of the preceding claims, wherein each oligonucleotide probe detects the largest possible number of different target nucleic acids resulting in maximum coverage for a given target nucleic acid population by the said library.

The library of oligonucleotide probes according to any one of the preceding claims, wherein the oligonucleotide probes are selected to have as many target sequences or binding sites as possible within the target population of nucleic acids in order to obtain a maximum degree of detection.

The library of oligonucleotide probes according to any one of the preceding claims, wherein the oligonucleotide probes are selected to have at least one target sequence in as many target nucleic acids as possible within the target population of nucleic acids in order to obtain a maximum degree of detection.

The library of oligonucleotide probes in TABLE 1 or TABLE 1a or FIG. 13 or FIG. 14 capable of detecting the complementary sequences in any given nucleic acid population.

The library according to any one of the preceding claims, which comprises probes each having a recognition element listed in TABLE 1 or TABLE 1a in the specification and/or which comprises probes each having a recognition element complementary to the recognition elements listed in said TABLE 1.

An oligonucleotide probe useful in a library according to any one of the preceding claims, said probe being selected from probes complementary to or identical with the sequences set forth in Table 1, FIG. 13, or FIG. 14.