Parallel sequencing of nucleic acid segments, useful for detecting single-nucleotide polymorphisms, by single-base extensions with labeled nucleotide

摘要

Parallel sequence analysis of segments of a complete nucleic acid sequence, using sequence-specific primers, is new. Parallel sequence analysis of segments of a complete nucleic acid sequence, using sequence-specific primers comprises: (i) preparing single-stranded nucleic acid chain fragments (X) of 30-1000 nucleotides (nt), corresponding to overlapping parts of the complete sequence; (ii) immobilizing (X) on a flat surface; (iii) hybridizing one or more sequence-specific primer populations to (X), where the density of individual extension-capable (X)-primer complexes is 10-1000 micron-2;and (iv) performing a cyclic 'build-up' reaction of strands complementary to (X), comprising: (a) treating immobilized (X) with a solution of at least one polymerase, 1-4 nucleotides (nt) labeled with a fluorophore (and if more than one nt is used the fluorophores on each can be differentiated) and modified so that the polymerase can add only one nt to the growing complementary strand, and the group that causes termination can be removed, along with the fluorophore; (b) incubating to extend each complementary strand by just one nt; (c) washing; (d) detecting, from fluorescence, the nt incorporated into each strand, and simultaneously the relative position on the reaction surface; (e) cleaving the terminating group and fluorophore from the complementary strands; (f) washing; and (g) repeating steps (a)-(f), optionally many times. The relative positions of individual (X) on the surface and their sequence are determined from the signals detected during step (d). An Independent claim is also included for a surface for immobilizing (X) randomly at 10-106 per 100 micron-2.