Method of library preparation avoiding the formation of adaptor dimers
First Claim
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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.
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Abstract
The invention relates to a method of preparing a library of template polynucleotides which reduces and/or prevents the formation of adaptor-dimers. The invention also relates to the use of a library of templates prepared using the method of the invention for solid-phase nucleic acid amplification. In particular, the invention relates to a method of preparing a library of template polynucleotides which have common sequences at their 5′ ends and at their 3′ ends which is substantially free of adaptor-dimers.
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Citations
13 Claims
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1. A method for generating a library of template polynucleotide molecules from one or more primary polynucleotide molecules, said method comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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Specification