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

• US 9,309,560 B2
• Filed: 05/19/2009
• Issued: 04/12/2016
• 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 R., Malek, Joel A., McKernan, Kevin J.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/468,818
• Publication Number:
  US 20100028888A1
• Time in Patent Office: 2,520 Days
• Field of Search: None
• US Class Current: 1/1
• 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 preparing a plurality of nucleic acids, comprising:

a) producing a plurality of circular nucleic acids, by intramolecularly ligating a plurality of nucleic acid fragments each having one or more first adaptors joined to the 5′ and

3′

ends of a linear nucleic acid fragment;

b) fragmenting the plurality of circularized nucleic acids by shearing, thereby producing a plurality of linear paired tag fragments containing the one or more first adaptors and a paired tag having the 5′

end and the 3′

end of the linear nucleic acid fragment in step (a); and

c) joining a second adapter to both ends of the plurality of linear paired tag fragments thereby producing a plurality of adaptor-joined paired tag fragments.

2. The method of claim 1, further comprising the step:

amplifying the plurality of adaptor-joined paired tag fragments thereby producing amplified paired tags.

3. The method of claim 2, further comprising:

sequencing the amplified paired tags.

4. The method of claim 2, wherein the plurality of adaptor-joined paired tag fragments are subjected to size fractionation prior to amplifying the plurality of adaptor-joined paired tag fragments.

5. The method hod of claim 1, further comprising the step:

sequencing the plurality of adaptor-joined paired tag fragments.

6. The method of claim 1, wherein the first adaptors include a 5′

overhang end, a 3′

overhang end or a blunt end.

7. The method of claim 1, wherein the first adaptors include a biotin moiety.

8. A method for characterizing a nucleic acid, comprising:

a) fragmenting a nucleic acid to produce a plurality of nucleic acid fragments, each nucleic acid fragment in the plurality having a 5′

end and a 3′

end;

b) joining one or more first adapters to the 5′ and

3′

ends of the plurality of nucleic acid fragments, thereby producing a plurality of first modified nucleic acid fragments;

c) intramolecularly ligating the plurality of modified nucleic acid fragments, thereby producing a plurality of circularized nucleic acids having the one or more first adapters located between the 5′

end and the 3′

end sequences of the nucleic acid fragment of step (a);

d) fragmenting the plurality of circularized nucleic acids by shearing, thereby producing a plurality of linear fragments containing the one or more first adaptors and a paired tag having the 5′

end and the 3′

end of the linear nucleic acid fragment in step (a); and

e) joining a second adapter to the 5′ and

3′

ends of the plurality of linear fragments thereby producing a plurality of second modified nucleic acid fragments.

9. The method of claim 8, further comprising:

amplifying the plurality of second modified nucleic acid fragments, thereby producing amplified paired tags.

10. The method of claim 9, further comprising:

sequencing the amplified paired tags.

11. The method of claim 8, further comprising:

sequencing the plurality of second modified nucleic acid fragments.

12. The method of claim 8, wherein the joining in step (b) is selected from a group consisting of ligation, annealing and recombination.

13. The method of claim 12, wherein the joining in step (b) comprises homologous recombination.

14. The method of claim 8, wherein the first adaptors include a 5′

overhang end, a 3′

overhang end or a blunt end.

15. The method of claim 8, wherein the first adaptors include a biotin moiety.

16. The method of claim 8, wherein the shearing comprises nebulization, hydrodynamic shearing through a small orifice, or sonication.

17. The method of claim 8, wherein the joining in step (e) comprises ligation.