Patent ID: 7582420

Claim:
A method for detecting at least 100 different target nucleic acid sequences of interest in a sample, each sequence comprising from 3′ to 5′, contiguous first, second, and third target domains, wherein the first target domain has a detection position one, two, three or four nucleotides from the 3′ terminal base of the second target domain, and the second target domain is at least one nucleotide, comprising the steps of: (a) providing a sample having at least 100 different target nucleic acid sequences of interest that are immobilized on a solid support; (b) contacting the sample with a set of probes for each of the at least 100 different target nucleic acid sequences of interest to form hybridization complexes, each set comprising: a first probe comprising from 5′ to 3′: a first universal priming sequence and a sequence that is substantially complementary to the first target domain and that has an interrogation position suitable for basepairing with the detection position; and a second probe comprising 5′ to 3′: a sequence substantially complementary to the third target domain, and a second universal priming sequence, wherein at least one probe contains a distinct adapter sequence not native to the target sequence of interest; (c) contacting the hybridization complexes with an extension enzyme and dNTPs, wherein for each hybridization complex, if the base at the interrogation position is perfectly complementary to the base at the detection position, then the first probe is extended along the second target domain; (d) ligating the extended first probes to second probes to form amplification templates; (e) amplifying the amplification templates with first and second universal primers to produce amplicons; (f) immobilizing the amplicons on solid phase capture probes that are specific to individual adapter sequences; and (g) detecting the presence of at least 100 different immobilized amplicons at the capture probes; thereby indicating of the presence of the at least 100 different target sequences of interest in the sample.