Method of preparing libraries of template polynucleotides
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.
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.
2. A method according to claim 1 wherein the initial primer extension reaction comprises annealing the single universal primer species to an adapter portion of each strand of each individual adapter-target construct and extending the primer species to form extension products complementary to each strand of each individual adapter-target construct.
3. A method according to claim 1 wherein the initial primer extension reaction comprises: a) annealing a single universal primer species to the mismatched regions of each of the adapter-target constructs, b) extending the primer species by sequential addition of nucleotides to form extension products complementary to at least one strand of each of the adapter-target constructs, and c) subjecting the products obtained in step b) to denaturing conditions, thereby separating the extension products from strands of the adapter-target constructs.
4. A method according to claim 3 wherein steps a) to c) are carried out once and the extension products collectively provide the library of different template polynucleotide molecules.
5. A method according to claim 3 wherein steps a) to c) are repeated one or more times and all the extension products collectively provide the library of different template polynucleotide molecules.
6. A method according to claim 1 wherein the initial primer extension reaction is carried out as part of a polymerase chain reaction and the amplification products of the PCR reaction collectively provide the library of different template polynucleotide molecules.
7. A method according to claim 1 wherein the mismatched adapter polynucleotides are forked adapters formed by annealing of partially complementary first and second polynucleotide strands, wherein a sequence of 5 or more consecutive nucleotides at 3′ end of the first strand is complementary to a sequence of 5 or more consecutive nucleotides the 5′ end of the second strand such that a double-stranded region of 5 or more consecutive base pairs is formed by annealing of the two strands and wherein a sequence of at least 10 consecutive nucleotides at the 5′ end of the first strand and a sequence of at least 10 consecutive nucleotides at the 3′ end of the second strand are not complementary such that an unmatched region of at least 10 consecutive nucleotides on each strand remains in single stranded form when the double-stranded region is annealed.
8. A method according to claim 7 wherein the double-stranded region formed when the two strands are annealed is from 5 to 20 consecutive base pairs in length.
9. A method according to claim 7 wherein the unmatched region consists of from 10 to 50 consecutive unpaired nucleotides on each strand.
10. A method according to claim 1 wherein the different target polynucleotide duplexes are fragments of genomic DNA.
11. A method according to claim 1 wherein the library of different template polynucleotide molecules comprises the whole genome of an individual.
12. A method according to claim 1 wherein the target polynucleotide duplexes are double-stranded cDNAs.
13. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises: preparing the library of different template polynucleotide molecules using the method according to claim 1 and carrying out a solid-phase nucleic acid amplification reaction wherein said library of different template polynucleotide molecules is amplified.
14. A method according to claim 13 wherein the library of different template polynucleotide molecules is amplified by solid-phase PCR using forward and reverse amplification primers.
15. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises: preparing the library of different template polynucleotide molecules using the method of claim 6 and carrying out a solid-phase nucleic acid amplification reaction, 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 primers identical to those used in preparing the library of different template polynucleotide molecules.
16. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises: preparing the library of different template polynucleotide molecules using the method of claim 6 and 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 primers which are not identical to those used in preparing the library of different template polynucleotide molecules.
17. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises: preparing the library of different template polynucleotide molecules using the method of claim 1 and 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 forward amplification primers capable of annealing to an adapter portion of the extension products formed in the initial primer extension reaction and reverse primers which are capable of annealing to extension products formed by extension of forward amplification primers annealed to the extension products formed in the initial primer extension reaction.
18. A method of using the library of different template polynucleotide molecules prepared according to the method of claim 1 , comprising amplifying the library of different template polynucleotide molecules by solid-phase PCR amplification.
19. A method of using the library of different template polynucleotide molecules prepared according to the method of claim 11 , comprising amplifying the library of different template polynucleotide molecules by whole genome amplification.
20. A method of whole genome amplification which comprises: preparing the library of different template polynucleotide molecules using the method according to claim 11 and carrying out a nucleic acid amplification reaction wherein the library of different template polynucleotide molecules are amplified.
21. The method of claim 1 , further comprising determining the sequence of at least a part of the different target polynucleotide duplexes in the library of different template polynucleotide molecules.
22. The method of claim 1 , wherein the mismatched adapter polynucleotides comprise a capture moiety.
23. The method of claim 22 , wherein the adapter-target constructs are attached to a surface by the capture moiety.
24. The method of claim 23 , further comprising a step of purifying the extension products from the adapter-target constructs.
25. The method of claim 1 , wherein the library of different template polynucleotides consists essentially of a whole genome from a single human individual.
26. The method of claim 6 , wherein the polymerase chain reaction is a whole genome polymerase chain reaction of genomic DNA from a human individual.
27. The method of claim 1 , wherein the single primer species comprises a non-hybridizing tail sequence at the 5′ end such that adapter target constructs are also extended.
28. The method of claim 1 , wherein the library of different template polynucleotide molecules comprises sequences of the whole complex polynucleotide sample.
29. A method of generating a library of different template polynucleotide molecules, the method comprising:
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; and
annealing 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.
30. The method of claim 29 , wherein the providing a plurality of different target polynucleotide duplexes comprises fragmenting a complex polynucleotide sample.