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 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.