Method of preparing libraries of template polynucleotides
First Claim
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1. A method of generating a library of different polynucleotide molecules, the method comprising:
- (a) providing a plurality of different target polynucleotide duplexes;
(b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region;
(c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and(d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs,wherein the extension products 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 polynucleotide adapters are forked adapters formed by annealing of partially complementary first and second polynucleotide strands; and
wherein a sequence of 5 or more consecutive nucleotides at 3′
end of the first strand of the forked adapters is complementary to a sequence of 5 or more consecutive nucleotides the 5′
end of the second strand of the forked adapters such that a double-stranded region of 5 or more consecutive base pairs is formed by annealing of the two strands.
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Abstract
The present invention relates to a method for preparing a library of template polynucleotides and use thereof in methods of solid-phase nucleic acid amplification. More specifically, the invention relates to a method for preparing a library of template polynucleotides that have common sequences at their 5′ ends and at their 3′ ends.
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Citations
25 Claims
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1. A method of generating a library of different polynucleotide molecules, the method comprising:
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(a) providing a plurality of different target polynucleotide duplexes; (b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region; (c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and (d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs, wherein the extension products 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 polynucleotide adapters are forked adapters formed by annealing of partially complementary first and second polynucleotide strands; and wherein a sequence of 5 or more consecutive nucleotides at 3′
end of the first strand of the forked adapters is complementary to a sequence of 5 or more consecutive nucleotides the 5′
end of the second strand of the forked adapters such that a double-stranded region of 5 or more consecutive base pairs is formed by annealing of the two strands. - View Dependent Claims (2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 16)
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6. A method of generating a library of different polynucleotide molecules, the method comprising:
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(a) providing a plurality of different target polynucleotide duplexes; (b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region; (c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and (d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs; wherein the extension products 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 single universal primer species comprises a non-hybridizing tail sequence at the 5′
end whereby the single primer species is extended to generate extension products that comprise the tail sequence or a complementary sequence thereof; andfurther comprising carrying out a solid-phase nucleic acid amplification reaction wherein the library of different template polynucleotide molecules is amplified, wherein the library of different template polynucleotide molecules is amplified by solid-phase PCR using forward and reverse amplification primers and solid-phase PCR is carried out using amplification primers capable of annealing to the tail sequence of the extension products or the complementary sequence thereof.
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14. A method of generating a library of different polynucleotide molecules, the method comprising:
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(a) providing a plurality of different target polynucleotide duplexes; (b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region; (c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and (d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs; wherein the extension products 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 library of different polynucleotide molecules is used to prepare a clustered array of nucleic acid colonies on a solid support. - View Dependent Claims (15)
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17. A method of generating a library of different polynucleotide molecules, the method comprising:
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(a) providing a plurality of different target polynucleotide duplexes; (b) providing identical polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched region; (c) ligating the double-stranded annealed regions of the identical polynucleotide adapters to both ends of the different target polynucleotide duplexes to form adapter-target constructs, and (d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs and extending the primer to form extension products complementary to both strands of the adapter-target constructs, wherein the extension products 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;further comprising carrying out a solid-phase nucleic acid amplification reaction wherein said library of different template polynucleotide molecules is amplified on a solid-phase surface; wherein the solid-phase surface is a solid support; and wherein the solid support is selected from the group consisting of latex beads, dextran beads, polystyrene, polypropylene surface, polyacrylamide gel, gold surfaces, glass surfaces and silicon wafers. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25)
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Specification