Multiplex analysis of polymorphic loci through simultaneous consultation and enzyme-mediated detection

摘要

Method (A) of simultaneous determination of nucleotide composition at designated polymorphic sites correlated located within one or more target nucleotide sequences, said method comprising the following steps: (a) providing one or more sets of probes, each probe being capable if one or more target nucleotide sequences located within a range of proximity to a first designated polymorphic site, where each probe comprises a terminal elongation initiation region capable of mating with the first designated polymorphic site, is paired with a sub-sequence of said , said terminal elongation initiation region comprising the 3-4 terminal nucleotides of the 3 'terminal end of the probe, and a duplex anchor region, and wherein the one or more sets of probes are immobilized on encoded microparticles, assembling the coded microparticles in a randomized coded alignment; (b) contacting the set of probes with said one or more target nucleotide sequences to allow the formation of hybridization complexes by placing a query site in the terminal elongation initiation sequence of a probe sequence in direct alignment with the first designated polymorphic site, in which the terminal elongation initiation region initiates a polymerase catalyzed elongation reaction to form an elongation product if said terminal elongation initiation sequence perfectly matches its corresponding site in the target nucleotide sequence, said corresponding site comprising the first designated polymorphic site, in which the polymerase lacks 3 '- 5' exonuclease activity; (c) contacting the elongation product with a second probe designed to hybridize with a second designated polymorphic site; (d) for each hybridization complex, determine the presence of the elongation product to indicate a coincidence or absence of the elongation product to indicate a mismatch between the consultation site and the first designated polymorphic site, and for each elongation product , determine the hybridization of the second probe with the second designated polymorphic site; and (e) determining the composition of the first and second correlated designated polymorphic sites, or (B) determining the composition of polymorphic sites in a target nucleic acid, said method comprising: providing a nucleic acid, said nucleic acid having polymorphic sites ; choose at least two polymorphic sites as designated sites; provide two or more probes capable of consulting the designated sites; wherein said probes comprise a terminal elongation initiation region located in the 3 'terminal position of the probe sequence; consulting the designated sites so as to establish the presence of two or more such designated sites and determining the compositions at said two or more sites, wherein the step of consulting the designated sites comprises forming a hybridization complex between a first probe and said nucleic acid and initiate a polymerase catalyzed elongation of the probe if said terminal elongation initiation region perfectly matches the corresponding portion of the target nucleic acid sequence in which the polymerase lacks 3 '- 5 exonuclease activity '; forming an elongation product by elongating said first probe; and forming a hybridization complex by pairing with the elongation product a second probe designed to consult a second designated site.