Method of preparing libraries of template polynucleotides

• US 20070128624A1
• Filed: 07/14/2006
• Published: 06/07/2007
• Est. Priority Date: 11/01/2005
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Gormley, Niall, Bentley, David, Rigatti, Roberto, Smith, Geoffrey
• Granted Patent: US 7,741,463 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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.

Claims

1. A method of generating a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends, the method comprising;

ligating identical mismatched adapter polynucleotides to both ends of each of one or more target polynucleotide duplexes to form one or more 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 an unmatched region, and

carrying out an initial primer extension reaction in which a primer oligonucleotide is annealed to an adapter portion 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 extension products, and optionally amplification products derived therefrom, collectively provide a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends.

2. A method according to claim 1 wherein the initial primer extension reaction comprises annealing an oligonucleotide primer to an adapter portion of each strand of each individual adapter-target construct and extending the primers 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 primer oligonucleotide to an adapter portion of each of the adapter-target constructs,

b) extending the primers 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 collected to provide a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends.

5. A method according to claim 3 wherein steps a) to c) are repeated one or more times and all the extension products collected to provide a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends.

6. A method according to claim 1 wherein the primers used in the initial primer extension reaction are annealed to an unmatched region of the adapters.

7. 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 are collected to provide a library of template polynucleotide molecules which have common sequences at their 5′

and 3′

ends.

8. A method according to claim 7 wherein the polymerase chain reaction is carried out using first oligonucleotide primers capable of annealing to adapter portions of each of the adapter-target constructs and second oligonucleotide primers capable of annealing to a region of the extended strands produced by extension of the first oligonucleotide primers, this region being complementary to an adapter portion of the adapter-target constructs.

9. A method according to claim 8 wherein the first and second oligonucleotide primers have different nucleotide sequences.

10. A method according to claim 9 wherein the first oligonucleotide primers are capable of annealing to the unmatched region in a first strand of the adapter-target constructs and the second oligonucleotide primers are capable of annealing to an extension product complementary to the unmatched region in a second strand of the adapter-target constructs.

11. A method according to claim 8 wherein the first and second oligonucleotide primers have substantially identical nucleotide sequences.

12. A method according to claim 11 wherein the first and second oligonucleotide primers are capable of annealing to a double-stranded region in the adapter portions of the adapter-target constructs.

13. 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.

14. A method according to claim 13 wherein the double-stranded region formed when the two strands are annealed is from 5 to 20 consecutive base pairs in length.

15. A method according to claim 13 wherein the unmatched region consists of from 10 to 50 consecutive unpaired nucleotides on each strand.

16. A method according to claim 1 wherein the target polynucleotide duplexes are fragments of genomic DNA.

17. A method according to claim 16 wherein the target polynucleotide duplexes are fragments of a whole genome.

18. A method according to claim 1 wherein the target polynucleotide duplexes are double-stranded cDNAs.

19. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises:

preparing a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends using the method according to claim 1 and carrying out a solid-phase nucleic acid amplification reaction wherein said template polynucleotide molecules are amplified.

20. A method according to claim 19 wherein said template polynucleotide molecules are amplified by solid-phase PCR using forward and reverse amplification primers.

21. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises:

preparing a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends using the method of claim 7 and carrying out a solid-phase nucleic acid amplification reaction wherein said template polynucleotide molecules are amplified, wherein said template polynucleotide molecules are 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 template polynucleotide molecules.

22. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises:

preparing a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends using the method of claim 7 and carrying out a solid-phase nucleic acid amplification reaction wherein said template polynucleotide molecules are amplified, wherein said template polynucleotide molecules are 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 template polynucleotide molecules.

23. A method of solid-phase nucleic acid amplification of template polynucleotide molecules which comprises:

preparing a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends using the method of claim 1 and carrying out a solid-phase nucleic acid amplification reaction wherein said template polynucleotide molecules are amplified, wherein said template polynucleotide molecules are 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.

24. A method of using a library of template polynucleotide molecules prepared according to the method of claim 1 as a template for solid-phase PCR amplification.

25. A method of using a library of template polynucleotide molecules prepared according to the method of claim 17 as a template for whole genome amplification.

26. A method of whole genome amplification which comprises:

preparing a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends using the method according to claim 17 and carrying out a nucleic acid amplification reaction wherein said template polynucleotide molecules are amplified.

27. A kit for use in preparing a library of template polynucleotide molecules which have common sequences at their 5′

ends and common sequences at their 3′

ends, the kit comprising mismatched adapter polynucleotides as defined in claim 1 and oligonucleotide primers capable of annealing to the mismatched adapter polynucleotides.

28. A kit according to claim 27 wherein the oligonucleotide primers are capable of annealing to an unmatched region of the mismatched adapter polynucleotides.

29. A kit according to claim 28 wherein the mismatched adapter polynucleotides are forked adapters.