Method of preparing libraries of template polynucleotides

• US 20160355880A1
• Filed: 06/03/2016
• Published: 12/08/2016
• Est. Priority Date: 11/01/2005
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Rigatti, Roberto, Gormley, Niall Anthony, Smith, Geoffrey Paul, Bentley, David, Luo, Shujun
• Granted Patent: US 10,253,359 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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. (canceled)

2. A forked polynucleotide adapter formed by annealing of partially complementary first and second polynucleotide strands, wherein at least one of the strands comprises a polynucleotide sequence complementary to the sequence of SEQ ID NO:

4.

3. The adapter of claim 2, wherein a sequence of at least 20 consecutive nucleotides at the 5′

end of the first strand and a sequence of at least 20 consecutive nucleotides at the 3′

end of the second strand are not complementary to each other, such that a mismatched region of at least 20 consecutive nucleotides on each strand remains in single stranded form when first and second strands are annealed.

4. The adapter of claim 2, wherein one of the first and second polynucleotide strands comprises the sequence of SEQ ID NO:

2.

5. A library of nucleic acids comprising a plurality of different polynucleotide duplexes having identical forked polynucleotide adapters at each end, wherein the adapters are formed by annealing of partially complementary first and second polynucleotide strands, and at least one of the strands comprises a polynucleotide sequence complementary to the sequence of SEQ ID NO:

4.

6. The library of claim 5, wherein a sequence of at least 20 consecutive nucleotides at the 5′

end of the first strand of the adapters and a sequence of at least 20 consecutive nucleotides at the 3′

end of the second strand of the adapters are not complementary to each other, such that a mismatched region of at least 20 consecutive nucleotides on each strand remains in single stranded form when the first and second strands are annealed.

7. The library of claim 5, wherein one of the first and second polynucleotide strands comprises the sequence of SEQ ID NO:

2.

8. The library of claim 5, wherein the library comprises sequences of a whole complex polynucleotide sample.

9. The library of claim 5, wherein the plurality of different polynucleotide duplexes are fragments of genomic DNA.

10. A method of generating a library of polynucleotide molecules, comprising:

(a) providing a plurality of different polynucleotide duplexes;

(b) providing identical forked polynucleotide adapters, wherein each adapter comprises a double-stranded annealed region and a mismatched single stranded region;

(c) ligating the double-stranded annealed regions of the adapters to both ends of the polynucleotide duplexes to form adapter-target constructs;

(d) annealing a single universal primer species to the mismatched regions of the adapter-target constructs; and

(e) extending the primer to form extension products complementary to both strands of the adapter-target constructs,

wherein the adapters are formed by annealing of partially complementary first and second polynucleotide strands, and at least one of the strands comprises a polynucleotide sequence complementary to the sequence of SEQ ID NO;

4.

11. The method of claim 10, wherein a sequence of at least 20 consecutive nucleotides at the 5′

end of the first strand and a sequence of at least 20 consecutive nucleotides at the 3′

end of the second strand are not complementary to each other, such that a mismatched region of at least 20 consecutive nucleotides on each strand remains in single stranded form when first and second strands are annealed.

12. The method of claim 10, wherein one of the first and second polynucleotide strands comprises the sequence of SEQ ID NO:

2.

13. The method of claim 10, further comprising carrying out a solid-phase nucleic acid amplification reaction, wherein the library of polynucleotide molecules is amplified on a solid support.

14. The method of claim 10, further comprising determining the sequence of at least a subset of the different polynucleotide duplexes.

15. The method of claim 10, further comprising purifying the extension products from the adapter-target constructs.

16. The method of claim 10, wherein the step of providing a plurality of different polynucleotide duplexes comprises fragmenting a complex polynucleotide sample.

17. The method of claim 16, wherein the library comprises sequences of the whole complex polynucleotide sample.

18. The method of claim 17, wherein the complex polynucleotide sample comprises genomic DNA.

19. The method of claim 13, wherein the amplification is carried out using a single surface bound primer.

20. The method of claim 13, further comprising carrying out a sequencing reaction to determine the sequence of at least a part of an amplified polynucleotide molecule.

21. The method of claim 13, wherein the solid support comprises a glass surface, a polyacrylamide gel, or latex beads.