METHODS FOR PRODUCING A PAIRED TAG FROM A NUCLEIC ACID SEQUENCE AND METHODS OF USE THEREOF

• US 20100028888A1
• Filed: 05/19/2009
• Published: 02/04/2010
• Est. Priority Date: 10/31/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Smith, Douglas R., McKernan, Kevin J., Malek, Joel A.
• Granted Patent: US 9,309,560 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-32. -32. (canceled)

33. A composition comprising nucleic acid sequence elements arranged in the following order:

linker 1—

5′

end tag—

linker 2—

3′

end tag—

linker 3

wherein the 5′

end tag and the 3′

end tag comprise a paired tag derived from a single contiguous nucleic acid sequence fragment.

34. (canceled)

35. The composition of claim 33, wherein linker 2 comprises:

a) at least two restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, and is oriented in such a way that one of the sites directs cleavage within the 5′

end tag or at the junction of linker 1 and the 5′

end tag, and the other site directs cleavage within the 3′

end tag or at the junction of linker 3 and the 3′

end tag; and

/or

b) at least one recognition site for a rare-cutting restriction endonuclease located between the two restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site.

36. The composition of claim 33, wherein linker 1 and linker 3 are the same sequence in reverse orientation.

37. The composition of claim 33, wherein the composition is amplified using oligonucleotides complementary to sequences present in linker 1 and linker 3.

38. The composition of claim 33, wherein linker 1 and linker 3 are derived by cleavage of a circular nucleic acid molecule with a rare-cutting restriction endonuclease comprising a recognition site between linker 1 and linker 3.

39. A composition comprising a circular nucleic acid molecule, wherein sequence elements are arranged in the following circular order:

40. (canceled)

41. The composition of claim 39, wherein linker 2 comprises:

a) at least two restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site, and are oriented in such a way that one of the sites directs cleavage within the 5′

end tag or at the junction of the 5′

end tag and linker 2 and, and the other site directs cleavage within the 3′

end tag or at the junction of the 3′

end tag and linker 2; and

/or

b) at least one recognition site for a rare-cutting restriction endonuclease located between the two restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site.

42. The composition of claim 39, wherein linker 2 is palindromic.

43. The composition of claim 39, wherein linker 2 comprises a recognition site for a rare-cutting restriction endonuclease.

44. The composition of claim 39, wherein the composition is amplified isothermally using oligonucleotide primers complementary to sequences in either linker 1 or linker 2, or in both linker 1 and linker 2.

45-81. -81. (canceled)