Parallel sequencing method

Abstract

A method for obtaining sequence information from a plurality of target polynucleotides in a sample is disclosed. In one embodiment, the method involves contacting a plurality of different sequence primers with a polynucleotide sample under conditions effective for the primers to anneal to primer-complementary regions in one or more target polynucleotides, to form one or more target-primer hybrid(s). Each different-sequence primer contains (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment. The hybrid(s) are contacted with a labeled nucleotide terminator in the presence of a primer-extending reagent, under conditions effective to append the base to an end of the annealed primer in the hybrid only when the base is complementary to a base in the target polynucleotide that is immediately adjacent to the end of the annealed primer, to form an extended hybrid mixture. The primers are then immobilized on an addressable array of immobilized, different tag complements, wherein each different tag complement contains a sequence that is complementary to the primer tag segments, under conditions effective to hybridize the primers to corresponding tag complements. The identification of a nucleotide base appended to at least one extended primer immobilized on the array allows determination of the presence of at least one target sequence in the sample.