Patent ID: 9328378
Filing Date: 2016-05-03
CPC Classification: C12N,C12Q

Claim Text:
1. A method for generating a library of template polynucleotide molecules from one or more primary polynucleotide molecules, said method comprising: (a) providing blunt end target polynucleotide duplexes; and performing a tailing reaction to attach a single nucleotide overhang to the 3′ ends of the blunt end target polynucleotide duplexes; (b) ligating an adaptor polynucleotide construct to both ends of the target polynucleotide duplexes to generate combined ligated adaptor-target-adaptor sequences; (c) preparing an amplification reaction comprising said combined ligated adaptor-target-adaptor sequences and at least two different primer oligonucleotides, wherein each of said at least two different primer oligonucleotides is complementary to at least a part of the adaptor polynucleotide construct sequence of the combined ligated adaptor-target-adaptor sequences, and complementary to the single nucleotide overhang attached to the target polynucleotide duplex, not extending beyond the single nucleotide overhang, and wherein a first primer of the two primer oligonucleotides is between 21-100 nucleotides in length and comprises a 5′ sequence having the sequence of nucleotides 1-21 of SEQ ID NO: 6; and (d) performing an amplification reaction wherein said at least two different primer oligonucleotides anneal to complementary parts of the adaptor-target-adaptor sequences and are extended by sequential addition of nucleotides to generate a plurality of amplification products complementary to at least one strand of the combined ligated adaptor-target-adaptor sequences, wherein each of said plurality of amplification products has a first common sequence at its 5′ end and a second common sequence at its 3′ end, and wherein said plurality of amplification products comprises a library of template polynucleotide molecules.