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

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.

1 - 3 . (canceled) 
     
     
         4 . A method for producing a paired tag from a first nucleic acid sequence fragment, without cloning, comprising the steps of:
 a) joining the 5′ and 3′ ends of a first nucleic acid sequence fragment to at least one adapter;   b) cleaving the adapter(s), thereby producing a second nucleic acid sequence fragment with compatible ends;   c) circularizing the second nucleic acid sequence fragment such that a 5′ end of the first nucleic acid sequence fragment is joined to a 3′ end of the first nucleic acid sequence fragment via a linker derived from the adapter(s), thereby producing a circular nucleic acid molecule;   d) cleaving the circular nucleic acid molecule, thereby producing 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 linker.   
     
     
         5 . The method of  claim 4 , wherein:
 a) the adapter comprises 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;   b) the adapter comprises a restriction endonuclease recognition site specific for a rare cutting restriction endonuclease;   c) cleaving the adapter joined to the nucleic acid sequence fragment with a rare cutting restriction endonuclease to produce the compatible ends; and   d) cleaving the circular nucleic acid molecule with a restriction endonuclease that cleaves distally to the restriction endonuclease recognition site.   
     
     
         6 . The method of  claim 5 , wherein the restriction endonuclease recognition sites specific for a restriction endonuclease that cleaves the nucleic acid sequence fragment distally to the restriction endonuclease recognition site are immediately adjacent to the 5′ and 3′ ends of the nucleic acid sequence fragment. 
     
     
         7 - 18 . (canceled) 
     
     
         19 . A method for characterizing a nucleic acid sequence, without cloning, comprising the steps of:
 a) fragmenting a nucleic acid sequence thereby producing a plurality of first nucleic acid sequence fragments having a 5′ end and a 3′ end;   b) joining the 5′ and 3′ ends of each first nucleic acid sequence fragment to a first linker such that the first linker is located between the 5′ end and the 3′ end of each first nucleic acid sequence fragment in a circular nucleic acid molecule;   c) cleaving the circular nucleic acid molecules, thereby producing a plurality of second nucleic acid sequence fragments wherein a subset of the fragments comprise a paired tag derived from each first nucleic acid sequence fragment joined via the first linker;   d) joining the 5′ and 3′ ends of each second nucleic acid sequence fragment via a second linker such that the second linker is located between the ends of each second nucleic acid sequence fragment to form a plurality of second circular nucleic acid molecules;   e) amplifying the second circular nucleic acid molecules two oligonucleotides complementary to sequences present in the second linker, thereby producing a plurality of amplified nucleic acid fragments; and   f) characterizing the 5′ and 3′ end tags of the plurality of amplified nucleic acid fragments.   
     
     
         20 - 24 . (canceled) 
     
     
         25 . The method of  claim 19 , wherein the second nucleic acid fragments are purified away from other nucleic acid fragments prior to PCR amplification. 
     
     
         26 . The method of  claim 25 , wherein the second nucleic acid fragment is purified from other nucleic acid fragments by affinity capture using a reagent specific for the first linker prior to amplification. 
     
     
         27 . The method of  claim 26 , wherein the first linker comprises a biotin moiety to enable affinity capture. 
     
     
         28 . The method of  claim 26 , wherein the first linker comprises a sequence capable of forming a triple helix to enable affinity capture. 
     
     
         29 . The method of  claim 26  in which the first linker comprises a recognition site for a DNA binding protein to enable affinity capture. 
     
     
         30 . The method of  claim 27 , wherein the nucleic acid sequence is a genome. 
     
     
         31 . The method of  claim 30 , wherein the method karyotypes the genome. 
     
     
         32 . The method of  claim 30 , wherein the method sequences the genome. 
     
     
         33 - 38 . (canceled) 
     
     
         39 . A composition comprising a circular nucleic acid molecule, wherein sequence elements are arranged in the following circular order: 
       
         
           
           
               
               
           
         
         wherein the 5′ end tag and the 3′ end tag comprise a paired tag derived from a single contiguous nucleic acid sequence fragment. 
       
     
     
         40 . The composition of  claim 39 , wherein linker 1 comprises 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 the other site directs cleavage within the 3′ end tag or at the junction of the 3′ end tag and linker 2. 
     
     
         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   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 . The composition of  claim 39 , wherein the composition is amplified isothermally using oligonucleotide primers complementary to sequences in either linker 1 or linker 2, and oriented in opposite directions such that a linear nucleic acid fragment is produced that preserves the orientation of the 5′ end tag and the 3′ end tag with respect to each other. 
     
     
         46 - 69 . (canceled)