Methods for producing a paired tag from a nucleic acid sequence and methods of use thereof

• US 9,822,395 B2
• Filed: 03/21/2016
• Issued: 11/21/2017
• Est. Priority Date: 10/31/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Smith, Douglas, Malek, Joel, McKernan, Kevin
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US15/075,742
• Publication Number:
  US 20160265034A1
• Time in Patent Office: 610 Days
• Field of Search: None
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

Methods for producing a paired tag from a nucleic acid sequence are provided in which the paired tag comprises the 5′ end tag and 3′ end tag of the nucleic acid sequence. In one embodiment, the nucleic acid sequence comprises two restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence distally to the restriction endonuclease recognition sites. In another embodiment, the nucleic acid sequence further comprises restriction endonuclease recognition sites specific for a rare cutting restriction endonuclease. Methods of using paired tags are also provided. In one embodiment, paired tags are used to characterize a nucleic acid sequence. In a particular embodiment, the nucleic acid sequence is a genome. In one embodiment, the characterization of a nucleic acid sequence is karyotyping. Alternatively, in another embodiment, the characterization of a nucleic acid sequence is mapping of the sequence. In a further embodiment, a method is provided for identifying nucleic acid sequences that encode at least two interacting proteins.

Claims

1. A method for producing a clonally amplified paired tag from a first nucleic acid sequence fragment, without cloning, comprising the steps of:

a) generating the first nucleic acid sequence fragment, wherein the first nucleic acid sequence fragment is joined to one or more first adapters at its 5′ and

3′

end;

b) performing an intramolecular ligation such that the one or more first adapters are located between the 5′

end and the 3′

end of the first nucleic acid sequence fragment in a circular nucleic acid molecule;

c) fragmenting the circular nucleic acid molecule, thereby producing a second nucleic acid fragment comprising a paired tag wherein a 5′

end tag of the first nucleic acid sequence fragment is joined to a 3′

end tag of the first nucleic acid sequence fragment via the one or more first adapters;

d) ligating one or more second adapters to the 5′ and

3′

ends of the second nucleic acid fragment, wherein at least one of the one or more second adapters comprise one or more primer binding sequences; and

e) clonally amplifying the second nucleic acid fragment using one or more primers that bind to the one or more primer binding sequences, thereby producing a clonally amplified paired tag.

2. The method of claim 1, wherein the second nucleic acid fragment is purified using affinity capture before ligating the one or more second adapters.

3. The method of claim 2, wherein at least one of the one or more first adapters comprise a biotin moiety and the affinity capture is performed using a streptavidin moiety.

4. The method of claim 1, wherein the clonally amplifying is performed using isothermal amplification.

5. The method of claim 1, wherein the circular nucleic acid molecule is fragmented by shearing.

6. The method of claim 1, wherein the circular nucleic acid molecule is fragmented by an endonuclease.

7. The method of claim 1, further comprising sequencing the clonally amplified paired tag.

8. The method of claim 7, wherein the sequencing is performed using sequencing-by-synthesis.

9. The method of claim 1, wherein the second nucleic acid fragment is immobilized to a substrate during the clonal amplification.

10. The method of claim 9, further comprising sequencing the immobilized second nucleic acid fragment wherein the sequencing is performed in both directions on the substrate.

11. The method of claim 9, wherein the substrate comprises at least one of the one or more primers immobilized thereon when clonally amplifying the second nucleic acid fragment.

12. The method of claim 1, wherein step a) comprises:

(i) fragmenting a nucleic acid sequence to produce a plurality of first nucleic acid sequence fragments each having a 5′

end and a 3′

end; and

(ii) joining the 5′ and

3′

ends of a first nucleic acid sequence fragment of the plurality of first nucleic acid sequence fragments to the one or more first adapters.

13. A method for characterizing a nucleic acid sequence, comprising:

a) generating first nucleic acid sequence fragments from the nucleic acid sequence, wherein the first nucleic acid sequence fragments comprise one or more first adapters on their 5′ and

3′

ends;

b) performing an intramolecular ligation such that the one or more first adapters are located between the 5′

ends and the 3′

ends of the first nucleic acid sequence fragments in a plurality of circular nucleic acid molecules;

c) fragmenting the plurality of circular nucleic acid molecules, thereby producing a plurality of second nucleic acid fragments each comprising a paired tag comprising a 5′

end tag joined to a 3′

end tag of a first nucleic acid sequence fragment via one or more of the one or more first adapters;

d) ligating one or more second adapters to the 5′ and

3′

ends of the plurality of second nucleic acid fragments, wherein at least one of the one or more second adapters comprise one or more primer binding sequences;

e) clonally amplifying the plurality of second nucleic acid fragments using one or more primers that bind to the one or more primer binding sequences, wherein the plurality of second nucleic acid fragments are immobilized on a substrate during the clonal amplification, wherein the substrate comprises at least one of the one or more primers immobilized thereto before the clonally amplifying; and

f) sequencing the clonally amplified plurality of second nucleic acid fragments, thereby characterizing the nucleic acid sequence.

14. The method of claim 13, wherein the plurality of second nucleic acid fragments are purified using affinity capture before ligating the one or more second adapters.

15. The method of claim 14, wherein at least one of the one or more first adapters comprise a biotin moiety and the affinity capture is performed using a streptavidin moiety.

16. The method of claim 13, wherein the clonally amplifying is performed using isothermal amplification.

17. The method of claim 13, wherein the plurality of circular nucleic acid molecules is fragmented by shearing.

18. The method of claim 13, wherein the plurality of circular nucleic acid molecules is fragmented by an endonuclease.

19. The method of claim 13, wherein the sequencing is performed using sequencing-by-synthesis.

20. The method of claim 13, wherein step a) comprises:

(i) fragmenting the nucleic acid sequence to produce a plurality of nucleic acid sequence fragments having a 5′

end and a 3′

end; and

(ii) joining the 5′ and

3′

ends of the nucleic acid sequence fragments to one or more first adapters.