Method of preparing libraries of template polynucleotides

Patent metadata

Field	Value
Publication number	US-11142789-B2
Application number	US-201916373433-A
Country	US
Kind code	B2
Filing date	Apr 2, 2019
Priority date	Nov 1, 2005
Publication date	Oct 12, 2021
Grant date	Oct 12, 2021

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.

First claim

The invention claimed is:

1. 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.

2. The method of claim 1 , 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.

3. The method of claim 1 , wherein one of the first and second polynucleotide strands comprises the sequence of SEQ ID NO: 2.

4. The method of claim 1 , further comprising carrying out a solid-phase nucleic acid amplification reaction, wherein the library of polynucleotide molecules is amplified on a solid support.

5. The method of claim 1 , further comprising determining the sequence of at least a subset of the different polynucleotide duplexes.

6. The method of claim 1 , further comprising purifying the extension products from the adapter-target constructs.

7. The method of claim 1 , wherein the step of providing a plurality of different polynucleotide duplexes comprises fragmenting a complex polynucleotide sample.

8. The method of claim 7 , wherein the library comprises sequences of the whole complex polynucleotide sample.

9. The method of claim 8 , wherein the complex polynucleotide sample comprises genomic DNA.

10. The method of claim 4 , wherein the amplification reaction is carried out using a single surface bound primer.

11. The method of claim 4 , further comprising carrying out a sequencing reaction to determine the sequence of at least a part of an amplified polynucleotide molecule.

12. The method of claim 4 , wherein the solid support comprises a glass surface, a polyacrylamide gel, or latex beads.

Assignees

• Illumina Cambridge Ltd

Inventors

• Gormley Niall Anthony
• Smith Geoffrey Paul
• Bentley David
• Rigatti Roberto
• Luo Shujun

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title

• C12Q1/6855 Primary

  Ligating adaptors · CPC title