Patent ID: 7741463
Filing Date: 2010-06-22
Classification: C12N,C12Q,C40B,Y10T

Abstract:
1. A method of generating a library of different template polynucleotide molecules, the method comprising: fragmenting a complex polynucleotide sample to generate a plurality of polynucleotide fragments, wherein the plurality of polynucleotide fragments are different target polynucleotide duplexes; ligating identical mismatched adapter polynucleotides to both ends of each of the different target polynucleotide duplexes to form adapter-target constructs, wherein each mismatched adapter is formed from two annealed polynucleotide strands that form a bimolecular complex comprising at least one double-stranded region and a mismatched region comprising portions of both strands, wherein the ligating covalently attaches each strand of the at least one double-stranded region to each respective strand of each of the different target polynucleotide duplexes to generate adapter-target constructs comprising covalently attached 5′ and 3′ adapter sequences; and carrying out an initial primer extension reaction in which a single universal primer species is annealed to the mismatched regions of each of the adapter-target constructs and extended by sequential addition of nucleotides to form extension products complementary to at least one strand of each of the adapter-target constructs, wherein the initial primer extension reaction of each of the adapter-target constructs generates extension products resulting from extension of the annealed single universal primer species that comprise nucleic acid sequences that differ from the nucleic acid sequences of either strand of the adapter-target constructs, and the extension products collectively provide a library of different template polynucleotide molecules which have common sequences at their 5′ ends and common sequences at their 3′ ends, wherein the common sequences at the 5′ ends of the extension products are not complementary to the common sequences at the 3′ ends of the extension products.