Patent ID: 6528288
Filing Date: 2003-03-04
Classification: C12Q,Y10T

Abstract:
A method of sequencing a nucleic acid template comprising:(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5â€² to or 3â€² to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the first plurality; (b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template; (c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; (d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments; (e) providing a plurality of second primers, each second primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5â€² to or 3â€² to the region affixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the second plurality; (f) repeating steps (b)-(d) for the second plurality of primers to thereby determine the nucleotide sequence of a second region of the template; and (g) assembling the first sequenced region and the second sequenced region of the template nucleic acid to form a first contig.