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Abstract

The present invention relates to methods for multiplex detection and quantification of target nucleic acid sequences in a sample comprising the steps of: (i) providing a solid support having immobilized thereon an array of detector oligonucleotides, wherein said array of detector oligonucleotides is designed by random selection of non-eukaryotic genomic sequences followed by random selection of oligonucleotide sequences and subsequent conversion of these oligonucleotide sequences such that these are composed of only three types of nucleotides; (ii) providing a sample having added thereto a fixed amount of control nucleic acid of known sequence; (iii) contacting said sample with at least two probes that hybridise to adjacent sites of a target sequence under conditions favouring hybridisation between the sample nucleic acids and the said at least two probes, wherein, a) a first probe is composed of a 5′ end sequence part for hybridisation to a PCR primer and a 3′ end sequence part for hybridisation to the target nucleic acid; and b) a second probe is composed of a 5′ end sequence part for hybridisation to the target nucleic acid, and a 3′ end sequence part for hybridisation with a PCR primer, and c) an intermediate sequence is present in between said 5′ and 3′ end sequence parts of said first or second probe; and d) said second probe is characterized by having 5′ phosphate group allowing ligation with a 3′ hydroxyl group at the said first probe forming a ligation-mediated probe; (iv) ligation of the said hybridised first and second probes to form ligation-mediated probes; (v) contacting a set of detectable labelled PCR primers with the ligation-mediated probes allowing amplification thereof; (vi) detection and quantification of sample nucleic acids via hybridisation of the said intermediate parts within the amplified ligation-mediated probes onto the array of detector oligonucleotides provided in The present invention also relates to the use of said methods as well as microarrays and kits for performing said methods.

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First Claim