Method of preparing libraries of template polynucleotides
First Claim
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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, andthe 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.
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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
30 Claims
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1. A method of generating a library of different template polynucleotide molecules, the method comprising:
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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; andcarrying 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. A method of generating a library of different template polynucleotide molecules, the method comprising:
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providing a plurality of different target polynucleotide duplexes; ligating identical double-stranded polynucleotide adapters to both ends of each of the different target polynucleotide duplexes to form adapter-target constructs, wherein each adapter comprises at least one double-stranded annealed region and a mismatched region comprising portions of both strands, wherein the ligating covalently attaches each strand of the at least one double-stranded annealed 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; andannealing a single universal primer species to the mismatched regions of each of the adapter-target constructs and performing an initial primer extension reaction by sequential addition of nucleotides to form extension products complementary to both strands of each of the adapter-target constructs, wherein the single 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, 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. - View Dependent Claims (30)
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Specification