End modification to prevent over-representation of fragments

• US 10,006,081 B2
• Filed: 03/24/2015
• Issued: 06/26/2018
• Est. Priority Date: 02/08/2006
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Rigatti, Roberto, Gormley, Niall Anthony, Bignell, Helen Rachel
• Primary Examiner(s): Bertagna, Angela M.
• Assistant Examiner(s): Oyeyemi, Olayinka A
• Application Number:
  US14/666,839
• Publication Number:
  US 20150197789A1
• Time in Patent Office: 1,190 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides and also the use of the 5′ and 3′ modified library of templates in methods of solid-phase nucleic acid amplification. In particular, the invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides which have common sequences at their 5′ ends and at their 3′ ends, wherein over-representation of “end” sequences of the primary polynucleotide molecules from when the 5′ and 3′ modified library is generated is greatly reduced or prevented.

Claims

1. A method of reducing representation of the ends of a primary polynucleotide sequence in a library of fragments generated from said primary polynucleotide sequence, the method comprising:

a) performing an amplification reaction with one or more modified amplification primers, said modified amplification primers comprising a modification configured to prevent fragments originating from the ends of the primary polynucleotide from ligating to an adapter, thereby forming a modified primary polynucleotide sequence;

b) fragmenting the modified primary polynucleotide sequence such that the 5′

terminal fragment and the 3′

terminal fragment each comprise the modification and the internal fragments do not comprise the modification;

c) ligating an adapter to the internal fragments generated in step b), thereby forming a library of ligated internal fragments generated from said primary polynucleotide sequence; and

d) optionally purifying the ligated internal fragments by removing the unligated terminal fragments.

2. The method of claim 1, wherein said modified amplification primers comprise a 5′

-amino or 5′

-biotin modification.

3. The method of claim 1, wherein said modified amplification primers comprise a modification that prevents nucleotide polymerase mediated copying of the full length of the primer.

4. The method of claim 3, wherein said modified amplification primers comprise an abasic site.

5. The method of claim 1, further comprising e) amplifying the ligated internal fragments generated in step c).

6. The method of claim 1, wherein the fragmenting of step (b) is done by sonication or nebulization.

7. The method of claim 1, wherein the primary polynucleotide sequence is a DNA molecule.

8. The method of claim 7, wherein the modified primary polynucleotide is generated by polymerase chain reaction.

9. The method of claim 8, wherein the modified primary polynucleotide is at least 5000 base pairs in length.

10. The method of claim 1, wherein the primary polynucleotide sequence is a genomic DNA fragment.

11. The method of claim 1, further comprising end-repairing and phosphorylating fragments generated in step b).

12. The method of claim 11, wherein the end-repairing and phosphorylating comprises treating the fragments generated in step b) with a polymerase and a polynucleotide kinase.

13. The method of claim 1, wherein the purifying comprises selectively capturing the ligated internal fragments.