Patent ID: 7361461

Claim:
A method for determination of amounts or relative proportions of more than one individual ribopolynucleotide sequence or subgroups thereof in a sample comprising a mixture of target ribopolynucleotide sequences using a quantitative affinity aided solution hybridization in combination with size- or mass-based fractionation for obtaining resolution, wherein the method comprises the consecutive steps of: (a) providing, one or more pools with more than one soluble polynucleotide probe, wherein each probe in said pool is complementary to an individual target ribopolynucleotide sequence in the sample, being present in a molar excess as compared to the target ribopolynucleotide sequences, and has approximately the same number of hybridizing nucleotides, which are complementary to said target ribopolynucleotide sequences, wherein approximately the same number of nucleotides means that the polynucleotide probes are not distinguishable from each other in size- or mass-based separation, fractionation and recording and are made distinguishable by providing said polynucleotide probes with one or more resolution enabling tags, which tags are oligonucleotide residues, which change the mass or size of the polynuclotide probes and provide them with different mobilities in fractionation, separation or recording systems without disturbing the hybridization or capturing reaction, wherein each pool of polynucleotide probes are placed in their own vessels; (b) providing a mixture of affinity tagged target ribopolynucleotide sequences by contacting the sample comprising the mixture of target ribopolynucleotide sequences with at least one affinity tag; and thereafter (c) performing steps (i) and (ii) simultaneously, or sequentially; in the order (i) and (ii), wherein steps (i) and (ii) comprise: (i) allowing a hybridization reaction to take place between the molar excess of polynucleotide probes from step (a) and the affinity tagged target ribopolynucleotide sequences from step (b) leading to formation of hybrids; (ii) providing captured hybrids by recovering the hybrids, on a separation aiding tool provided with an affinity pair of the affinity tag of the target ribopolynucleotide sequences; (d) providing released polynucleotide probes by eluting the polynucleotide probes in an unmodified form from the captured hybrids, wherein said released polynucleotide probes are provided with tracer tags in step (a) or are tracer tagged after release in step (d) or during or after amplification after the release in step (d); (e) separating the released polynucleotide probes by electrophoretic or chromatographic techniques or mass spectrometry and recording the amount or relative proportions of distinguishable polynucleotide probes, the amount of which corresponds to the amount of complementary target ribopolynucleotide sequences in the mixture of target ribopolynucleotide sequences in the sample.