Multiplexed analysis of polymorphic loci by simultaneous consultation and enzyme-mediated detection

摘要

Method of simultaneous determination of nucleotide composition at designated polymorphic sites located within one or more target nucleotide sequences, said target nucleotide sequences comprising an undesignated polymorphism, said method comprising the following steps: (a) providing a set of pairs of oligonucleotide primers, each pair being able to mate with complementary polynucleotide chains to delineate a corresponding target region that includes at least one designated polymorphic site; (b) contacting said set of oligonucleotide primers with said targets under conditions that allow the formation of amplicons with designated polymorphic sites corresponding to the designated polymorphic sites on corresponding targets, each amplicon comprising a sense amplicon chain corresponding to a target sense chain and an antisense amplicon chain corresponding to a target antisense chain; (c) providing one or more sets of degenerate probes, in which the set contains degenerate probes capable of mating with a sequence of said one or more sequences of sense chain and amplicon antisense chain located within a range of proximity to a site polymorphic designated in the sense or antisense chain; (d) contacting the set of probes with said one or more amplicon nucleotide sequences to allow the formation of hybridization complexes by placing a query site within a probe sequence in direct alignment with the designated polymorphic site in the chain. sense and antisense; (e) incubating each hybridization complex with a DNA polymerase capable of extending the probes within the complex: (i) determining the presence of a match between the consultation site and a designated polymorphic site by probe elongation; and (ii) determine the presence of a mismatch between the consultation site and a polymorphic site designated by the lack of probe elongation; thereby determining the composition of the designated polymorphic site; wherein the one or more sets of probes are immobilized on encoded microparticles, the encoded microparticles being assembled in a randomly coded alignment.